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The oxidative pentose-phosphate pathway (OPPP) retrieves NADPH from glucose-6-phosphate, which is important in chloroplasts at night and in plastids of heterotrophic tissues. We previously studied how OPPP enzymes may transiently locate to peroxisomes, but how this is achieved for the third enzyme remained unclear. By extending our genetic approach, we demonstrated that Arabidopsis isoform 6-phosphogluconate dehydrogenase 2 (PGD2) is indispensable in peroxisomes during fertilization, and investigated why all PGD-reporter fusions show a mostly cytosolic pattern. A previously published interaction of a plant PGD with thioredoxin m was confirmed using Trxm2 for yeast two-hybrid (Y2H) and bimolecular fluorescent complementation (BiFC) assays, and medial reporter fusions (with both ends accessible) proved to be beneficial for studying peroxisomal targeting of PGD2. Of special importance were phosphomimetic changes at Thr6, resulting in a clear targeting switch to peroxisomes, while a similar change at position Ser7 in PGD1 conferred plastid import. Apparently, efficient subcellular localization can be achieved by activating an unknown kinase, either early after or during translation. N-terminal phosphorylation of PGD2 interfered with dimerization in the cytosol, thus allowing accessibility of the C-terminal peroxisomal targeting signal (PTS1). Notably, we identified amino acid positions that are conserved among plant PGD homologues, with PTS1 motifs first appearing in ferns, suggesting a functional link to fertilization during the evolution of seed plants.

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OBJECTIVE: Secreted phospholipase A2 Group IB (sPLA2GIB) regulates the release of arachidonic acid, prostaglandins, and other inflammatory lipid mediators. Although it has been well involved in extensive inflammatory diseases, its specific mechanism in chronic rhinosinusitis with nasal polyps (CRSwNP) remains unclear. In this study, we investigated the role of sPLA2GIB in the pathophysiology of CRSwNP. METHODS: Quantitative PCR, immunofluorescence staining, western blotting, and enzyme-linked immunosorbent assay (ELISA) were used to analyze the expression of sPLA2s, phospholipase A2 receptor (PLA2R), and prostaglandin D2 (PGD2) in nasal samples. Human nasal epithelial cells (HNECs) were cultured at an air-liquid interface (ALI) and stimulated with various cytokines. The human mast cell line HMC-1 was stimulated with sPLA2GIB, and the expression of PGD2 and cytokines in the culture supernatant was detected by ELISA. RESULTS: The mRNA and protein levels of sPLA2GIB were significantly higher in eosinophilic CRSwNP than in control tissues. sPLA2GIB was predominantly expressed in the nasal epithelial cells. PLA2R mRNA and protein levels were upregulated in both eosinophilic and non-eosinophilic CRSwNP compared with the control groups. IL-4, IL-13, TNF-α, and IL-1ß upregulated the expression of sPLA2GIB in ALI-cultured HNECs. sPLA2GIB induced PGD2 and IL-13 production in HMC-1 cells in a hydrolytic activity-independent manner. PGD2 protein expression was elevated in tissue homogenates of eosinophilic CRSwNP, and PGD2 upregulated the expression of IL-13 in HMC-1 cells. CONCLUSION: Increased secretion of sPLA2GIB by epithelial cells may promote eosinophilic inflammation in CRSwNP by enhancing PGD2 and IL-13 production in mast cells via binding to PLA2R. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:1107-1117, 2024.

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BACKGROUND: Rosmarinic acid (RosA) is a natural phenolic compound that possesses a wide-range of pharmacological properties. However, the effects of RosA on influenza A virus-mediated acute lung injury remain unknown. In this study, we aimed to explore whether RosA could protect against H1N1 virus-mediated lung injury and elucidate the underlying mechanisms. METHODS: Mice were intragastrically administered with RosA for 2 days before intranasal inoculation of the H1N1 virus (5LD50) for the establishment of an acute lung injury model. At day 7 post-infection (p.i.), gross anatomic lung pathology, lung histopathologic, and lung index (lung weight/body weight) were examined. Luminex assay, multiple immunofluorescence and flow cytometry were performed to detect the levels of pro-inflammatory cytokines and apoptosis, respectively. Western blotting and plasmid transfection with hematopoietic-type PGD2 synthase (h-PGDS) overexpression were conducted to elucidate the mechanisms. RESULTS: RosA effectively attenuated H1N1 virus-triggered deterioration of gross anatomical morphology, worsened lung histopathology, and elevated lung index. Excessive pro-inflammatory reactions, aberrant alveolar epithelial cell apoptosis, and cytotoxic CD8+ T lung recruitment in the lung tissues induced by H1N1 virus infection were observed to be reduced by RosA treatment. In vitro experiments demonstrated that RosA treatment dose-dependently suppressed the increased levels of pro-inflammatory mediators and apoptosis through inhibition of nuclear factor kappa B (NF-κB) and P38 MAPK signaling pathways in H1N1 virus-infected A549 cells, which was accompanied by promoting activation of the h-PGDS-PGD2-HO-1 signal axis. Furthermore, we strikingly found that h-PGDS inhibition significantly abrogated the inhibitory effects of RosA on H1N1 virus-mediated activation of NF-κB and P38 MAPK signaling pathways, resulting in diminishing the suppressive effects on the increased levels of pro-inflammatory cytokines and chemokines as well as apoptosis. Finally, suppressing h-PGDS prominently abolished the protective effects of RosA on H1N1 virus-mediated severe pneumonia and lung injury. CONCLUSIONS: Taken together, our study demonstrates that RosA is a promising compound to alleviate H1N1 virus-induced severe lung injury through prompting the h-PGDS-PGD2-HO-1 signal axis.

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Recently, a G-protein coupled receptor 44 (GPR44) was discovered to play a significant role in the process of inflammation-related diseases, including cancer and diabetes. However, the precise role of GPR44 has yet to be fully elucidated. Currently, there is a strong and urgent need for the development of GPR44 radiotracers as a non-invasive methodology to explore the exact mechanism of GPR44 on inflammation-related diseases and monitor the progress of therapy. TM-30089 is a potent GPR44 antagonist that exhibits a high specificity and selectivity for GPR44. Its structure contains a fluorine nuclide, which could potentially be replaced with 18F. In the present study, we successfully took a highly effective synthesis strategy that pretreated the unprotected carboxylic acid group of the precursor and developed a feasible one-step automatic radiosynthesis strategy for [18F]TM-30089 with a high radiochemical purity and a good radiochemical yield. We further evaluated this radiotracer using mice models implanted with 1.1 B4 cell lines (GPR44-enriched cell lines) and human islets (high GPR44 expression), respectively. The results revealed the persistent and specific uptake of [18F]TM-30089 in GPR44 region, indicating that [18F]TM-30089 is a promising candidate for targeting GPR44. Further evaluation is ongoing.

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BACKGROUND: Prostaglandin D2 (PGD2) has been shown to restrict the occurrence and development of multiple cancers; nevertheless, its underlying molecular mechanism has not been fully elucidated. The present study investigated the effect of PGD2 on the biological function of the enriched gastric cancer stem cells (GCSCs), as well as its underlying molecular mechanism, to provide a theoretical basis and potential therapeutic drugs for gastric cancer (GC) treatment. METHODS: The plasma PGD2 levels were detected by Enzyme-linked immunosorbent assay (ELISA). Silencing of lipocalin prostaglandin D synthetases (L-PTGDS) and prostaglandin D2 receptor 2 (PTGDR2) was carried out in GCSCs from SGC-7901 and HGC-27 cell lines. Cell Counting Kit-8, transwell, flow cytometry, and western blotting assays were used to determine cell viability, invasion, apoptosis, and stemness of GCSCs. In vivo xenograft models were used to assess tumor growth. RESULTS: Clinically, it was found that the plasma PGD2 level decreased significantly in patients with GC. PGD2 suppressed viability, invasion, and stemness and increased the apoptosis of GCSCs. Downregulating L-PTGDS and PTGDR2 promoted viability, invasion, and stemness and reduced the apoptosis of GCSCs. Moreover, the inhibition of GCSCs induced by PGD2 was eliminated by downregulating the expression of PTGDR2. The results of in vivo experiments were consistent with those of in vitro experiments. CONCLUSION: Our data suggest that PGD2 may be an important marker and potential therapeutic target in the clinical management of GC. L-PTGDS/PTGDR2 may be one of the critical targets for GC therapy. The PGD2/PTGDR2 signal affects the viability, invasion, apoptosis, and stemness of GCSCs and prevents the progression of GC.

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Arachidonic acid-derived prostaglandins are widely studied for their role in inflammation. However, besides arachidonic acid, other arachidonic moiety-containing lipids can be metabolized by COX-2. Indeed, the endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide, AEA) can follow the same biochemical pathways than arachidonic acid leading to the formation of prostaglandin-glycerol esters (PG-G) and prostaglandin-ethanolamides (or prostamides, PG-EA), respectively. The data reported so far support the interest of these bioactive lipids in inflammatory conditions. However, there is only a handful of methods described for their quantification in biological matrices. Moreover, given the shared biochemical pathways for arachidonic acid, 2-AG and AEA, a method allowing for the quantification of these precursors and the corresponding prostaglandin derivatives appears as largely needed. Thus, we report here the development and validation of a single run UPLC-MS/MS quantification method allowing the quantification of these endocannabinoids-derived mediators together with the classical prostaglandin. Moreover, we applied the method to the quantification of these lipids in vitro (using lipopolysaccharides-activated J774 macrophage cells) and in vivo in several tissues from DSS-induced colitis mice. This femtomole-range method should improve the understanding of the interaction between these lipid mediators and inflammation.

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Nonsteroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (N-ERD) is characterized by nasal polyp formation, adult-onset asthma, and hypersensitivity to all cyclooxygenase-1 (COX-1) inhibitors. Oxygenated lipids are collectively known as oxylipins and are polyunsaturated fatty acids (PUFA) oxidation products. The most extensively researched oxylipins being the eicosanoids formed from arachidonic acid (AA). There are four major classes of eicosanoids including leukotrienes, prostaglandins, thromboxanes, and lipoxins. In N-ERD, the underlying inflammatory process of the upper and lower respiratory systems begins and occurs independently of NSAID consumption and is due to the overproduction of cysteinyl leukotrienes. Leukotriene mediators all induce edema, bronchoconstriction, and airway mucous secretion. Thromboxane A2 is a potent bronchoconstrictor and induces endothelial adhesion molecule expression. Elevated Prostaglandin D2 metabolites lead to vasoconstriction, additionally impaired up-regulation of prostaglandin E2 leads to symptoms seen in N-ERD as it is essential for maintaining homeostasis of inflammatory responses in the airway and has bronchoprotective and anti-inflammatory effects. A characteristic feature of N-ERD is diminished lipoxin levels, this decreased capacity to form endogenous mediators with anti-inflammatory properties could facilitate local inflammatory response and expose bronchial smooth muscle to relatively unopposed actions of broncho-constricting substances. Treatment options, such as leukotriene modifying agents, aspirin desensitization, biologic agents and ESS, appear to influence eicosanoid pathways, however more studies need to be done to further understand the role of oxylipins. Besides AA-derived eicosanoids, other oxylipins may also pay a role but have not been sufficiently studied. Identifying pathogenic N-ERD mechanism is likely to define more effective treatment targets.

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Prostaglandin D2 (PGD2) is one of the most potent endogenous sleep-promoting molecules. However, the cellular and molecular mechanisms of the PGD2-induced activation of sleep-promoting neurons in the ventrolateral preoptic nucleus (VLPO), the major nonrapid eye movement (NREM)-sleep center, still remains unclear. We here show that PGD2 receptors (DP1) are not only expressed in the leptomeninges but also in astrocytes from the VLPO. We further demonstrate, by performing real-time measurements of extracellular adenosine using purine enzymatic biosensors in the VLPO, that PGD2 application causes a 40% increase in adenosine level, via an astroglial release. Measurements of vasodilatory responses and electrophysiological recordings finally reveal that, in response to PGD2 application, adenosine release induces an A2AR-mediated dilatation of blood vessels and activation of VLPO sleep-promoting neurons. Altogether, our results unravel the PGD2 signaling pathway in the VLPO, controlling local blood flow and sleep-promoting neurons, via astrocyte-derived adenosine.

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We previously reported that the addition of prostaglandin, (PG)D2, and its chemically stable analog, 11-deoxy-11-methylene-PGD2 (11d-11m-PGD2), during the maturation phase of 3T3-L1 cells promotes adipogenesis. In the present study, we aimed to elucidate the effects of the addition of PGD2 or 11d-11m-PGD2 to 3T3-L1 cells during the differentiation phase on adipogenesis. We found that both PGD2 and 11d-11m-PGD2 suppressed adipogenesis through the downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) expression. However, the latter suppressed adipogenesis more potently than PGD2, most likely because of its higher resistance to spontaneous transformation into PGJ2 derivatives. In addition, this anti-adipogenic effect was attenuated by the coexistence of an IP receptor agonist, suggesting that the effect depends on the intensity of the signaling from the IP receptor. The D-prostanoid receptors 1 (DP1) and 2 (DP2, also known as a chemoattractant receptor-homologous molecule expressed on Th2 cells) are receptors for PGD2. The inhibitory effects of PGD2 and 11d-11m-PGD2 on adipogenesis were slightly attenuated by a DP2 agonist. Furthermore, the addition of PGD2 and 11d-11m-PGD2 during the differentiation phase reduced the DP1 and DP2 expression during the maturation phase. Overall, these results indicated that the addition of PGD2 or 11d-11m-PGD2 during the differentiation phase suppresses adipogenesis via the dysfunction of DP1 and DP2. Therefore, unidentified receptor(s) for both molecules may be involved in the suppression of adipogenesis.

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The number of people suffering from hair loss is increasing, and hair loss occurs not only in older men but also in women and young people. Prostaglandin D2 (PGD2) is a well-known alopecia inducer. However, the mechanism by which PGD2 induces alopecia is poorly understood. In this study, we characterized CXXC5, a negative regulator of the Wnt/ß-catenin pathway, as a mediator for hair loss by PGD2. The hair loss by PGD2 was restored by Cxxc5 knock-out or treatment of protein transduction domain-Dishevelled binding motif (PTD-DBM), a peptide activating the Wnt/ß-catenin pathway via interference with the Dishevelled (Dvl) binding function of CXXC5. In addition, suppression of neogenic hair growth by PGD2 was also overcome by PTD-DBM treatment or Cxxc5 knock-out as shown by the wound-induced hair neogenesis (WIHN) model. Moreover, we found that CXXC5 also mediates DHT-induced hair loss via PGD2. DHT-induced hair loss was alleviated by inhibition of both GSK-3ß and CXXC5 functions. Overall, CXXC5 mediates the hair loss by the DHT-PGD2 axis through suppression of Wnt/ß-catenin signaling.

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Dairy cows often develop different degrees of endometritis after calving and this is attributed to pathogenic bacterial infections such as by Escherichia coli and Staphylococcus aureus. Infection of the bovine endometrium causes tissue damage and increases the expression of prostaglandin D2 (PGD2), which exerts anti-inflammatory effects on lung inflammation. However, the roles of PGD2 and its DP1 receptor in endometritis in cows remain unclear. Here, we examined the anti-inflammatory roles of the lipocalin-type prostaglandin D2 synthase (L-PGDS)/PGD2 and DP1 receptor regulatory pathways in bovine endometritis. We evaluated the regulatory effects of PGD2 on inflammation and tissue damage in E. coli- and S. aureus-infected bovine endometrial cells cultured in vitro. We found that the secretion of pro-inflammatory cytokines interleukin (IL)-6, IL-1ß, and tumour necrosis factor (TNF)-α as well as expression of matrix metalloproteinase (MMP)-2, platelet-activating factor receptor (PAFR), and high mobility group box (HMGB)-1 were suppressed after DP1 receptor agonist treatment. In contrast, IL-6, IL-1ß, and TNF-α release and MMP-2, PAFR, and HMGB-1 expression levels were increased after treatment of bovine endometrial tissue with DP1 receptor antagonists. DP1-induced anti-inflammatory effects were dependent on cellular signal transduction. The L-PGDS/PGD2 pathway and DP1 receptor induced anti-inflammatory effects in bovine endometrium infected with S. aureus and E. coli by inhibiting the mitogen-activated protein kinase and nuclear factor-κB signalling pathways, thereby reducing tissue damage. Overall, our findings provide important insights into the pathophysiological roles of PGD2 in bovine endometritis and establish a theoretical basis for applying prostaglandins or non-steroidal anti-inflammatory drugs for treating endometrial inflammatory infertility in bovines.

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Inhibition of microsomal prostaglandin E synthase-1 (mPGES-1) results in decreased production of proinflammatory PGE2 and can lead to shunting of PGH2 into the prostaglandin D2 (PGD2)/15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) pathway. 15dPGJ2 forms Michael adducts with thiol-containing biomolecules such as GSH or cysteine residues on target proteins and is thought to promote resolution of inflammation. We aimed to elucidate the biosynthesis and metabolism of 15dPGJ2 via conjugation with GSH, to form 15dPGJ2-glutathione (15dPGJ2-GS) and 15dPGJ2-cysteine (15dPGJ2-Cys) conjugates and to characterize the effects of mPGES-1 inhibition on the PGD2/15dPGJ2 pathway in mouse and human immune cells. Our results demonstrate the formation of PGD2, 15dPGJ2, 15dPGJ2-GS, and 15dPGJ2-Cys in RAW264.7 cells after lipopolysaccharide stimulation. Moreover, 15dPGJ2-Cys was found in lipopolysaccharide-activated primary murine macrophages as well as in human mast cells following stimulation of the IgE-receptor. Our results also suggest that the microsomal glutathione S-transferase 3 is essential for the formation of 15dPGJ2 conjugates. In contrast to inhibition of cyclooxygenase, which leads to blockage of the PGD2/15dPGJ2 pathway, we found that inhibition of mPGES-1 preserves PGD2 and its metabolites. Collectively, this study highlights the formation of 15dPGJ2-GS and 15dPGJ2-Cys in mouse and human immune cells, the involvement of microsomal glutathione S-transferase 3 in their biosynthesis, and their unchanged formation following inhibition of mPGES-1. The results encourage further research on their roles as bioactive lipid mediators.

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Fevipiprant is a non-steroidal oral prostaglandin D2 (PGD2) receptor 2 antagonist that reduces bronchial wall inflammation, possibly improving clinical outcomes in the asthmatic population. A systemic review search was conducted on PubMed, Embase, and Central Cochrane Registry. Randomized clinical trials were included with Fevipiprant as an intervention arm compared to placebo. For continuous variables, the standardized mean difference, and for discrete variables, Mantel-Haenszel Risk Ratio (MH Risk ratio) was used for analysis. Confidence interval of 95% and p-value < 0.05 was considered significant. The analysis was done using a random-effects model irrespective of heterogeneity. Heterogeneity was evaluated using the I2 statistic. A total of five articles, including seven trials, were included in the analysis. There was significant increase in post-bronchodilator forced expiratory volume in one second (FEV1) 0.249 (0.157-0.341), p<0.001 and pre-bronchodilator FEV1 0.115 (0.043 to 0.188), p=0.002. A decrease in asthma control questionnaire (ACQ) score of -0.124 (-0.187 to -0.062), p<0.001, was reported. Statistically significant asthma exacerbation reduction was reported in the high eosinophil count population with a daily dose of 450mg 0.77 relative risks (RR) (0.61-0.97). There was a positive deviation toward Fevipiprant 450mg dose for asthma reduction in the overall population, but it was not statistically significant. Fevipiprant produced a slight statistically significant reduction in asthma exacerbations in the high eosinophil count population with favorable deviation in the overall population. It significantly increased pre-and post-bronchodilator FEV1 and improved ACQ scores in treated patients. The benefits, though statistically significant, failed to translate into clinical importance.

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Systemic lupus erythematosus is a complex autoimmune disease during which patients develop autoantibodies raised against nuclear antigens. During the course of the disease, by accumulating in secondary lymphoid organs (SLOs), basophils support autoreactive plasma cells to amplify autoantibody production. We have recently shown that murine lupus-like disease could be controlled by 10 days of oral treatment with a combination of prostaglandin D2 (PGD2) receptor (PTGDR) antagonists through the inhibition of basophil activation and recruitment to SLOs. Importantly, inhibiting solely PTGDR-1 or PTGDR-2 was ineffective, and the development of lupus-like disease could only be dampened by using antagonists for both PTGDR-1 and PTGDR-2. Here, we aimed at establishing a proof of concept that a clinically relevant bispecific antagonist of PTGDR-1 and PTGDR-2 could be efficient to treat murine lupus-like nephritis. Diseased Lyn-deficient female mice received treatment with AMG853 (vidupiprant, a bispecific PTGDR-1/PTGDR-2 antagonist) for 10 days. This led to the dampening of basophil activation and recruitment in SLOs and was associated with a decrease in plasmablast expansion and immunoglobulin E (IgE) production. Ten days of treatment with AMG853 was consequently sufficient in reducing the dsDNA-specific IgG titers, circulating immune complex glomerular deposition, and renal inflammation, which are hallmarks of lupus-like disease. Thus, bispecific PTGDR-1 and PTGDR-2 antagonists, such as AMG853, are a promising class of drugs for the treatment or prevention of organ damage in systemic lupus erythematosus.

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We previously reported that prostaglandin (PG)D2 and its isosteric analog, 11-deoxy-11-methylene-PGD2 (11d-11m-PGD2), promote adipogenesis in 3T3-L1 cells during the maturation phase. Focusing on the differentiation phase, although both PGs inhibited adipogenesis, this effect was canceled out by PGI2 and PGJ2 derivatives. Thus, PGD2 and 11d-11m-PGD2 play different roles during the phases, but do not affect PGI2- and PGJ2-derivative-induced adipogenesis.

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Tuft cells are a type of intestinal epithelial cells that exist in epithelial barriers and play a critical role in immunity against parasite infection. It remains insufficiently clear whether Tuft cells participate in bacterial eradication. Here, we identified Sh2d6 as a signature marker for CD45+ Tuft-2 cells. Depletion of Tuft-2 cells resulted in susceptibility to bacterial infection. Tuft-2 cells quickly expanded in response to bacterial infection and sensed the bacterial metabolite N-undecanoylglycine through vomeronasal receptor Vmn2r26. Mechanistically, Vmn2r26 engaged with N-undecanoylglycine activated G-protein-coupled receptor-phospholipase C gamma2 (GPCR-PLCγ2)-Ca2+ signaling axis, which initiated prostaglandin D2 (PGD2) production. PGD2 enhanced the mucus secretion of goblet cells and induced antibacterial immunity. Moreover, Vmn2r26 signaling also promoted SpiB transcription factor expression, which is responsible for Tuft-2 cell development and expansion in response to bacterial challenge. Our findings reveal an additional function of Tuft-2 cells in immunity against bacterial infection through Vmn2r26-mediated recognition of bacterial metabolites.

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BACKGROUND/AIMS: Despite significant advances in diagnostic and operative techniques, lung cancer remains one of the most lethal malignancies worldwide. Since prostaglandins such as prostaglandin D2 (PGD2) is involved in various pathophysiological process, including inflammation and tumorigenesis, this study aims to investigate the role of PGD2 during the process of epithelial-mesenchymal transition (EMT) in A549 cells. METHODS: A549 cells were stimulated with PGD2 and expression of EMT markers was analyzed by immunoblotting and immunofluorescence. EMT-related gene, Slug expression was evaluated using quantitative real-time polymerase chain reaction (qPCR). Migration and invasion abilities of A549 cells were determined in chemotaxis and Matrigel invasion assays, respectively. We also inhibited the TGF/Smad signaling pathway using a receptor inhibitor or silencing of TGF-ß1 and TGFß type I receptor (TGFßRI), and protein expression was assessed by immunoblotting and immunofluorescence. RESULTS: Here, we found that stimulation of A549 cells with PGD2 resulted in morphological changes into a mesenchymal-like phenotype under low serum conditions. Stimulation of A549 cells with PGD2 resulted in a significant reduction in proliferation, whereas invasion and migration were enhanced. The expression of E-cadherin was markedly downregulated, while Vimentin expression was upregulated after treatment of A549 cells with PGD2. Slug expression was markedly upregulated by stimulating A549 cells with PGD2, and stimulation of A549 cells with PGD2 significantly enhanced TGF-ß1 expression, and silencing of TGF-ß1 significantly blocked PGD2-induced EMT and Smad2 phosphorylation. In addition, PGD2-induced Smad2 phosphorylation and EMT were significantly abrogated by either pharmacological inhibition or silencing of TGFßRI. PGD2-induced expression of Slug and EMT were significantly augmented in low nutrient and low serum conditions. Finally, the subsequent culture of mesenchymal type of A549 cells under normal culture conditions reverted the cell's phenotype to an epithelial type. CONCLUSION: Given these results, we suggest that tumor microenvironmental factors such as PGD2, nutrition, and growth factors could be possible therapeutic targets for treating metastatic cancers.

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BACKGROUND: There are clinically relevant sex differences in acute and chronic pain mechanisms, but we are only beginning to understand their mechanistic basis. Transcriptome analyses of rodent whole dorsal root ganglion (DRG) have revealed sex differences, mostly in immune cells. We examined the transcriptome and translatome of the mouse DRG with the goal of identifying sex differences. METHODS: We used translating ribosome affinity purification sequencing and behavioral pharmacology to test the hypothesis that in Nav1.8-positive neurons, most of which are nociceptors, translatomes would differ by sex. RESULTS: We found 80 genes with sex differential expression in the whole DRG transcriptome and 66 genes whose messenger RNAs were sex differentially actively translated (translatome). We also identified different motifs in the 3' untranslated region of messenger RNAs that were sex differentially translated. In further validation studies, we focused on Ptgds, which was increased in the translatome of female mice. The messenger RNA encodes the prostaglandin PGD2 synthesizing enzyme. We observed increased PTGDS protein and PGD2 in female mouse DRG. The PTGDS inhibitor AT-56 caused intense pain behaviors in male mice but was only effective at high doses in female mice. Conversely, female mice responded more robustly to another major prostaglandin, PGE2, than did male mice. PTGDS protein expression was also higher in female cortical neurons, suggesting that DRG findings may be generalizable to other nervous system structures. CONCLUSIONS: Our results demonstrate sex differences in nociceptor-enriched translatomes and reveal unexpected sex differences in one of the oldest known nociceptive signaling molecule families, the prostaglandins.

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Large epidemiological studies often require sample transportation and storage, presenting unique considerations when applying advanced lipidomics techniques. The goal of this study was to acquire lipidomics data on plasma and serum samples stored at potential preanalytical conditions (e.g., thawing, extracting, evaporating), systematically monitoring lipid species for a period of one month. Split aliquots of 10 plasma samples and 10 serum samples from healthy individuals were kept in three temperature-related environments: refrigerator, laboratory benchtop, or heated incubator. Samples were analyzed at six different time points over 28 days using a Bligh & Dyer lipid extraction protocol followed by direct infusion into a lipidomics platform using differential mobility with tandem mass spectrometry. The observed concentration changes over time were evaluated relative to method and inter-individual biological variability. In addition, to evaluate the effect of lipase enzyme levels on concentration changes during storage, we compared corresponding fasting and post-prandial plasma samples collected from 5 individuals. Based on our data, a series of low abundance free fatty acid (FFA), diacylglycerol (DAG), and cholesteryl ester (CE) species were identified as potential analytical markers for degradation. These FFA and DAG species are typically produced by endogenous lipases from numerous triacylglycerols (TAGs), and certain high abundance phosphatidylcholines (PCs). The low concentration CEs, which appeared to increase several fold, were likely mass-isobars from oxidation of other high concentration CEs. Although the concentration changes of the high abundant TAG, PC, and CE precursors remained within method variability, the concentration trends of FFA, DAG, and oxidized CE products should be systematically monitored over time to inform analysts about possible pre-analytical biases due to degradation in the study sample sets.