scholarly journals Inactivation Effect of Thymoquinone on Alicyclobacillus acidoterrestris Vegetative Cells, Spores, and Biofilms

2021 ◽  
Vol 12 ◽  
Author(s):  
Qiuxia Fan ◽  
Cheng Liu ◽  
Zhenpeng Gao ◽  
Zhongqiu Hu ◽  
Zhouli Wang ◽  
...  
Keyword(s):  
Nigella Sativa ◽  
Membrane Integrity ◽  
Dependent Manner ◽  
Active Components ◽  
Concentration Dependent Manner ◽  
Vegetative Cells ◽  
Alicyclobacillus Acidoterrestris ◽  
Beverage Industry ◽  
Main Challenge ◽  
Bacterial Cell Morphology

Alicyclobacillus acidoterrestris (A. acidoterrestris), a spore-forming bacterium, has become a main challenge and concern for the juices and acid beverage industry across the world due to its thermo-acidophilic characteristic. Thymoquinone (TQ) is one of the active components derived from Nigella sativa seeds. The objective of this study was to investigate antibacterial activity and associated molecular mechanism of TQ against A. acidoterrestris vegetative cells, and to evaluate effects of TQ on A. acidoterrestris spores and biofilms formed on polystyrene and stainless steel surfaces. Minimum inhibitory concentrations of TQ against five tested A. acidoterrestris strains ranged from 32 to 64 μg/mL. TQ could destroy bacterial cell morphology and membrane integrity in a concentration-dependent manner. Field-emission scanning electron microscopy observation showed that TQ caused abnormal morphology of spores and thus exerted a killing effect on spores. Moreover, TQ was effective in inactivating and removing A. acidoterrestris mature biofilms. These findings indicated that TQ is promising as a new alternative to control A. acidoterrestris and thereby reduce associated contamination and deterioration in the juice and acid beverage industry.

Nigella sativa L., Supplementary Plant with Anticholinesterase Effect for Cognition Problems: A Kinetic Study

2020 ◽  
Vol 13 (2) ◽  
pp. 129-135 ◽  
Author(s):  
Mehdi Ansari ◽  
Ali Mandegary ◽  
Niloofar Mosalanejhad ◽  
Amir Asadi ◽  
Fariba Sharififar
Keyword(s):  
Kinetic Study ◽  
Nigella Sativa ◽  
The Elderly ◽  
Dependent Manner ◽  
Ache Inhibition ◽  
Concentration Dependent Manner ◽  
Total Extract ◽  
Ache Inhibitors ◽  
Main Component

Background: The average lifespan and the aging population are rising worldwide. So Neurodegenerative Disease (ND) will be one of the most common challenges associated with this population and would be more prevalent in future. The use of Acetylcholinesterase (AChE) inhibitors is one of the most important strategies for memory impairment. Medicinal plants are the most known natural source for accessing the new therapeutic agents. Objective: In this work, we aimed to study in vitro anticholinesterase effect of different concentrations (10, 100, 250, 500, 750 and 1000 μg/ml) of total extract of N. sativa (NTE) and its separated fractions and to study the kinetic of AChE enzyme in the presence of two concentrations of NTE (10 and 100 μg/ml). Methods: Maceration method was used for NTE preparation and different fractions of Petroleum Ether (PTE), Chloroform (CHF) and Methanol (MF). NTE, fractions and the main component of the plant, Thymoquinone (TQ), were assayed for AChE inhibition, using Ellman’s method. Kinetic study of the AChE enzyme was studied in the presence of NTE at 10 and 100 μg/ml using Linweaver- Burk plot too. Results: NTE and all the separated fractions inhibited AChE enzyme in a concentration-dependent manner. The greatest inhibition was shown by CHF and PEF fractions (86.97% and 79.99% at 1000 μg/ml, respectively). With less intensity, NTE, TQ and MF exhibited 76.32%, 68.98 % and 48.39% enzyme inhibition at 1000μg/ml, respectively. The least IC50 value was due to CHF fraction in AChE inhibition (98.28 ± 6.74 μg/ml). Kinetic profile exhibited the mixed mode of AChE inhibition by NTE. This indicates that a particular substance could not be responsible for AChE inhibition, and probably a collection of phytochemicals are involved in this process. Conclusions: N. sativa is a good candidate for seeking the new anticholinesterase agent and could be considered as a good supplement for the health of the elderly.

scholarly journals Haplopine Ameliorates 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis-Like Skin Lesions in Mice and TNF-α/IFN-γ-Induced Inflammation in Human Keratinocyte

Antioxidants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 806
Author(s):  
Tae-Young Kim ◽  
Ye Jin Kim ◽  
Jonghwan Jegal ◽  
Beom-Geun Jo ◽  
Han-Seok Choi ◽  
...  
Keyword(s):  
Atopic Dermatitis ◽  
Skin Lesions ◽  
Skin Thickness ◽  
Dependent Manner ◽  
Active Components ◽  
Concentration Dependent Manner ◽  
Gm Csf ◽  
Tnf Α ◽  
Protein Expressions

This study aimed to investigate the anti-inflammatory, antioxidant, and anti-atopic dermatitis (AD) effects of haplopine, which is one of the active components in D. dasycarpus. Haplopine (12.5 and 25 mM) inhibited the mRNA expressions of inflammatory cytokines IL-6, TSLP, GM-CSF, and G-CSF and the protein expressions of IL-6 and GM-CSF in TNF-α/INF-γ-stimulated HaCaT cells. In H2O2-induced Jukat T cells, haplopine (25 and 50 mM) suppressed the productions of proinflammatory cytokines (IL-4, IL-13, and COX-2) and increased the mRNA and protein expressions of oxidative stress defense enzymes (SOD, CAT, and HO-1) in a concentration-dependent manner. In vivo, haplopine significantly attenuated the development of AD symptoms in 2,4-dinitrochlorobenzene (DNCB)-stimulated Balb/c mice, as evidenced by reduced clinical dermatitis scores, skin thickness measurements, mast cell infiltration, and serum IgE concentrations. These findings demonstrate that haplopine should be considered a novel anti-atopic agent with the potential to treat AD.

Celastrol ameliorates acetaminophen-induced oxidative stress and cytotoxicity in HepG2 cells

2017 ◽  
Vol 37 (7) ◽  
pp. 742-751 ◽  
Author(s):  
AT Jannuzzi ◽  
M Kara ◽  
B Alpertunga
Keyword(s):  
Oxidative Stress ◽  
Cell Viability ◽  
Hepg2 Cells ◽  
Membrane Integrity ◽  
Neutral Red ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Tripterygium Wilfordii Hook F ◽  
Cell Membrane Integrity ◽  
And Oxidative Stress

Acetaminophen (APAP) is the most commonly used analgesic and antipyretic drug in the world. However, hepatotoxicity caused by APAP overdose is the most frequent cause of acute liver failure worldwide and oxidative stress involved in the pathogenesis of APAP hepatotoxicity. Celastrol is a natural triterpenoid derived from Tripterygium wilfordii Hook F. that exhibits antioxidant, anti-inflammatory, and antitumor activities. In this study, we aimed to investigate the potential ameliorative effects of celastrol against APAP-induced cytotoxicity and oxidative stress. Human hepatocellular carcinoma cells (HepG2) were incubated with 20 mM of APAP for 24 h and posttreated with 50 nM, 100 nM, or 200 nM of celastrol for a further 24 h. The methylthiazolyldiphenyl-tetrazolium bromide, lactate dehydrogenase, and neutral red uptake assays showed celastrol posttreatments recovered cell viability and cell membrane integrity in a concentration-dependent manner. Celastrol posttreatments exerted a significant increase in the glutathione content and a decrease in the malondialdehyde and protein carbonylation levels. Also, celastrol posttreatments attenuated the APAP-induced oxidative stress by raising glutathione peroxidase, glutathione reductase, and catalase activities. However, superoxide dismutase activity did not change. In conclusion, celastrol treatment may improve cell viability and increase cellular antioxidant defense in HepG2 cells. These results suggest that celastrol may have the potential to ameliorate the APAP-induced oxidative stress and cytotoxicity.

scholarly journals Characterization of Phospholipase A2 Enzyme Activity in Serum of the Komodo Dragon (Varanus komodoensis)Soluble phospholipase A2 (sPLA2) is an enzyme found in the peripheral circulation of vertebrates which has significant immunological activity. This en

2018 ◽  
Vol 11 (1) ◽  
pp. 2163-2169 ◽  
Author(s):  
Mark Merchant ◽  
Bekky Muscher-Hodges ◽  
Judith Bryja
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Phospholipase A2 ◽  
Membrane Integrity ◽  
Peripheral Circulation ◽  
Linear Increase ◽  
Dependent Manner ◽  
Immunological Activity ◽  
Concentration Dependent Manner ◽  
Minute Period

Soluble phospholipase A2 (sPLA2) is an enzyme found in the peripheral circulation of vertebrates which has significant immunological activity.  This enzyme exerts immune activity by the hydrolysis of fatty acids from the sn-2 position of membrane glycerophospholipids of microbes, thus compromising membrane integrity and casing eventual lysis.  We utilized membrane fatty acids labeled with a fluorescent probe (BODIPY) at the sn-2 position fatty acid to label the membranes.  Incubation of different volumes of serum from Komodo dragons with BODIPY-labeled bacteria resulted in liberation of labeled fatty acid in a serum volume-dependent manner. This cleavage of fatty acid occurred rapidly, with a biphasic production of fluorescent product.  An immediate accumulation of product was noted, which increased steadily for a 30-minute period, followed by a slower hydrolysis between 30 and 180 min.  The activity was temperature-dependent, with low activities observed at 5oC and a linear increase up to 40oC.  The liberation for fatty acid was inhibited by p-bromophenacyl bromide, a specific phospholipase A2 (PLA2) inhibitor, in a concentration-dependent manner, indicating that the activity was due to the presence of sPLA2.

Basement membrane integrity and keratinization in healthy and ulcerated bovine hoof tissue

2003 ◽  
Vol 70 (1) ◽  
pp. 19-27 ◽  
Author(s):  
Kay AK Hendry ◽  
Christopher H Knight ◽  
Hugh Galbraith ◽  
Colin J Wilde
Keyword(s):  
Basement Membrane ◽  
Dna Synthesis ◽  
Membrane Integrity ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Membrane Disruption ◽  
Dependent Manner ◽  
Antibody Treatment ◽  
Concentration Dependent Manner ◽  
Membrane Breakdown ◽  
Bovine Hoof

Damage to, or deterioration of, the keratinized horn tissue of the bovine hoof claw culminates ultimately in the development of solear ulceration. We have observed abnormal keratin distribution at the site of solear ulceration in the bovine claw that may be due to alteration of the positional cues of the keratinocytes. In this study we have characterized key cell biological changes associated with ulceration in the claw that may precipitate abnormal keratinization. Loss of basement membrane at sites of ulceration was found by immunofluorescent detection of laminin and integrins. In other tissues, basement membrane breakdown results from degradation by matrix metalloproteinases (MMPs). Similarly, elevated levels of MMPs 2 and 9 were observed in ulcerated bovine claw tissue both by zymography and, quantitatively, by assay of enzyme activity. In the sole of claws that contained an ulcer, tissue distal to the ulcer site also had elevated MMP 2 when compared with healthy sole tissue from the same animals, as did sole tissue of claws recovering from ulceration. Tissue inhibitor of metalloproteinase 2 (TIMP 2) was detected by ELISA in healthy tissue. TIMP 2 tended to be lower in diseased tissue distal to ulcer sites, and was significantly lower in ulcerated tissue. MMP 2 was located by immunofluorescence in the dermal and basal epidermal region of sole tissue, in the region of the basement membrane. Increased punctate staining of material in the dermis was associated with ulcerated material. ELISA of TIMP 2 in tissue extracts enriched for dermis or epidermis confirmed that the inhibitor was located predominantly in the dermis. To investigate a possible causal relationship between basement membrane anchorage and epidermal keratinization, the effect of function-blocking antibodies to laminins and integrins was tested in tissue explant cultures prepared from healthy sole tissue. Anti-integrin antibody treatment had no effect on either protein or DNA synthesis. In contrast, in the presence of anti-laminin antibody, protein synthesis was decreased in a concentration-dependent manner, a significant effect being observed at the highest concentration after treatment for 24 h. At this concentration, DNA synthesis was also decreased after 48 h of culture, an effect that may be relevant to a hibernal reduction in claw cell turnover, and the associated seasonal vulnerability of cows to claw damage. The results provide evidence for basement membrane disruption at ulcer sites, and an increased potential for disruption in the diseased claw, and a causal link between this and abnormal epidermal keratinization. Basement membrane disruption is in turn associated with reciprocal changes in MMPs and their inhibitors, favouring extracellular proteolysis. Whether MMP activation is the primary cause of dermal–epidermal deterioration and, if so, how MMP activation is triggered, remains to be determined.

scholarly journals The LiaFSR System Regulates the Cell Envelope Stress Response in Streptococcus mutans

2009 ◽  
Vol 191 (9) ◽  
pp. 2973-2984 ◽  
Author(s):  
Prashanth Suntharalingam ◽  
M. D. Senadheera ◽  
Richard W. Mair ◽  
Céline M. Lévesque ◽  
Dennis G. Cvitkovitch
Keyword(s):  
Stress Response ◽  
Streptococcus Mutans ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Transcriptional Analysis ◽  
Dependent Manner ◽  
Bacterial Survival ◽  
Concentration Dependent Manner ◽  
Lipid Ii ◽  
Envelope Stress

ABSTRACT Maintaining cell envelope integrity is critical for bacterial survival, including bacteria living in a complex and dynamic environment such as the human oral cavity. Streptococcus mutans, a major etiological agent of dental caries, uses two-component signal transduction systems (TCSTSs) to monitor and respond to various environmental stimuli. Previous studies have shown that the LiaSR TCSTS in S. mutans regulates virulence traits such as acid tolerance and biofilm formation. Although not examined in streptococci, homologs of LiaSR are widely disseminated in Firmicutes and function as part of the cell envelope stress response network. We describe here liaSR and its upstream liaF gene in the cell envelope stress tolerance of S. mutans strain UA159. Transcriptional analysis established liaSR as part of the pentacistronic liaFSR-ppiB-pnpB operon. A survey of cell envelope antimicrobials revealed that mutants deficient in one or all of the liaFSR genes were susceptible to Lipid II cycle interfering antibiotics and to chemicals that perturbed the cell membrane integrity. These compounds induced liaR transcription in a concentration-dependent manner. Notably, under bacitracin stress conditions, the LiaFSR signaling system was shown to induce transcription of several genes involved in membrane protein synthesis, peptidoglycan biosynthesis, envelope chaperone/proteases, and transcriptional regulators. In the absence of an inducer such as bacitracin, LiaF repressed LiaR-regulated expression, whereas supplementing cultures with bacitracin resulted in derepression of liaSR. While LiaF appears to be an integral component of the LiaSR signaling cascade, taken collectively, we report a novel role for LiaFSR in sensing cell envelope stress and preserving envelope integrity in S. mutans.

ALLELOPATHIC EFFECT OF β-CEMBRENEDIOL AND ITS MODE OF ACTION: INDUCED OXIDATIVE STRESS IN LETTUCE SEEDLINGS

2017 ◽  
pp. 441 ◽  
Author(s):  
Xia Ren ◽  
Zhi-qiang Yan ◽  
Xiao-feng He ◽  
Xiu-zhuang Li ◽  
Bo Qin
Keyword(s):  
Oxidative Stress ◽  
Lipid Peroxidation ◽  
Mode Of Action ◽  
Membrane Integrity ◽  
Shoot Elongation ◽  
Allelopathic Effect ◽  
Dependent Manner ◽  
Oxygen Species ◽  
Fresh Weight ◽  
Concentration Dependent Manner

β-cembrenediol((1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol), shown to be one of the most important allelochemicals of tobacco in our previous studies, effectively inhibited the root and shoot elongation of receptor plants in a concentration-dependent manner. However, its mechanism of action remains unclear. Seedlings of lettuce (Lactuca sativa) were now treated with β-cembrenediol to clarify its mode of action. Results showed that β-cembrenediol significantly inhibited the seeding growth and reduced fresh weight of L. saiva. The compounds effectively arrested cell division and caused cell death. Exposure to β-cembrenediol induced overproduction of reactive oxygen species. Moreover, increased levels of malondialdehyde, proline and hydrogen peroxide, and decreased in chlorophyll content indicated lipid peroxidation and induction of oxidative stress. These results suggested that β-cembrenediol caused oxidative damage through enhanced generation of ROS, as indicated by increased lipid peroxidation, disruption of membrane integrity and impacted mitosis, ultimately resulted in growth inhibition of the receptor plant.

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak
Keyword(s):  
Neutrophil Elastase ◽  
Cell Activation ◽  
Thromboxane A2 ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

Trimucytin: A Collagen-Like Aggregating Inducer Isolated from Trimeresurus mucrosquamatus Snake Venom

1993 ◽  
Vol 69 (03) ◽  
pp. 286-292 ◽  
Author(s):  
Che-Ming Teng ◽  
Feng-Nien Ko ◽  
Inn-Ho Tsai ◽  
Man-Ling Hung ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Aggregation ◽  
Snake Venom ◽  
Inositol Phosphate ◽  
Shape Change ◽  
Platelet Activating Factor ◽  
Atp Release ◽  
Platelet Membrane ◽  
Thromboxane B2 ◽  
Dependent Manner ◽  
Concentration Dependent Manner

SummaryTrimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytinand collagen-induced platelet aggregation and ATP release. However, trimucytin did not replace the binding of 125I-labeled MAB1988 to platelets. Platelets pre-exposed to trimucytin were resistant to the second challenge with trimucytin itself or collagen. It is concluded that trimucytin may activate collagen receptors on platelet membrane, and cause aggregation and release mainly through phospholipase C-phosphoinositide pathway.

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