Bioactive lipid mediators in plasma are predictors of preeclampsia irrespective of aspirin therapy.

There are few early biomarkers to identify pregnancies at risk of preeclampsia (PE) and abnormal placental function. In this cross-sectional study, we utilized targeted ultra-performance liquid chromatography-ESI MS/MS and a linear regression model to identify specific bioactive lipids that serve as early predictors of PE. Plasma samples were collected from 57 pregnant women prior to 24-weeks of gestation with outcomes of either PE (n = 26) or uncomplicated term pregnancies (n = 31), and the profiles of eicosanoids and sphingolipids were evaluated. Significant differences were revealed in the eicosanoid, (±)11,12 DHET, as well as multiple classes of sphingolipids; ceramides, ceramide-1-phosphate, sphingomyelin, and monohexosylceramides; all of which were associated with the subsequent development of PE regardless of aspirin therapy. Profiles of these bioactive lipids were found to vary based on self-designated race. Additional analyses demonstrated that PE patients can be stratified based on the lipid profile as to PE with a preterm birth linked to significant differences in the levels of 12-HETE, 15-HETE, and resolvin D1. Furthermore, subjects referred to a high-risk OB/GYN clinic had higher levels of 20-HETE, arachidonic acid, and Resolvin D1 versus subjects recruited from a routine, general OB/GYN clinic. Overall, this study shows that quantitative changes in plasma bioactive lipids detected by ultra-performance liquid chromatography-ESI-MS/MS can serve as an early predictor of PE and stratify pregnant people for PE type and risk.

RNA splicing variants of the novel long non-coding RNA, CyKILR, possess divergent biological functions in non-small cell lung cancer.

The CDKN2A gene, responsible for encoding the tumor suppressors p16(INK4A) and p14(ARF), is frequently inactivated in non-small cell lung cancer (NSCLC). In this study, an uncharacterized long non-coding RNA (lncRNA) (ENSG00000267053) on chromosome 19p13.12 was found to be overexpressed in NSCLC cells with an active CDKN2A gene. This lncRNA, named Cy clin-Dependent K inase I nhibitor 2A-regulated l nc R NA (CyKILR), also correlated with the STK11 gene-coded tumor suppressor Liver kinase B1 (LKB1). CyKILR displayed two splice variants, CyKILRa (with exon 3) and CyKILRb (without exon 3), which are synergistically regulated by CDKN2A and STK11 as knockdown of both tumor suppressor genes led to a significant loss of exon 3 inclusion in mature CyKILR RNA. CyKILRa localized to the nucleus, and its downregulation using antisense RNA oligonucleotides enhanced cellular proliferation, migration, clonogenic survival, and tumor incidence. In contrast, CyKILRb localized to the cytoplasm, and downregulation of CyKILRb using siRNA reduced cell proliferation, migration, clonogenic survival, and tumor incidence. Transcriptomics analyses revealed enhancement of apoptotic pathways with concomitant suppression of key cell cycle pathways by CyKILRa demonstrating its tumor-suppressive role, while CyKILRb inhibited tumor suppressor microRNAs, indicating an oncogenic nature. These findings elucidate the intricate roles of lncRNAs in cell signaling and tumorigenesis.

Ceramide-1-phosphate is a regulator of Golgi structure and is co-opted by the obligate intracellular bacterial pathogen Anaplasma phagocytophilum.

Many intracellular pathogens structurally disrupt the Golgi apparatus as an evolutionarily conserved promicrobial strategy. Yet, the host factors and signaling processes involved are often poorly understood, particularly for Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis. We found that A. phagocytophilum elevated cellular levels of the bioactive sphingolipid, ceramide-1-phosphate (C1P), to promote Golgi fragmentation that enables bacterial proliferation, conversion from its non-infectious to infectious form, and productive infection. A. phagocytophilum poorly infected mice deficient in ceramide kinase, the Golgi-localized enzyme responsible for C1P biosynthesis. C1P regulated Golgi morphology via activation of a PKCα/Cdc42/JNK signaling axis that culminates in phosphorylation of Golgi structural proteins, GRASP55 and GRASP65. siRNA-mediated depletion of Cdc42 blocked A. phagocytophilum from altering Golgi morphology, which impaired anterograde trafficking of trans-Golgi vesicles into and maturation of the pathogen-occupied vacuole. Cells overexpressing phosphorylation-resistant versions of GRASP55 and GRASP65 presented with suppressed C1P- and A. phagocytophilum-induced Golgi fragmentation and poorly supported infection by the bacterium. By studying A. phagocytophilum, we identify C1P as a regulator of Golgi structure and a host factor that is relevant to disease progression associated with Golgi fragmentation.IMPORTANCECeramide-1-phosphate (C1P), a bioactive sphingolipid that regulates diverse processes vital to mammalian physiology, is linked to disease states such as cancer, inflammation, and wound healing. By studying the obligate intracellular bacterium Anaplasma phagocytophilum, we discovered that C1P is a major regulator of Golgi morphology. A. phagocytophilum elevated C1P levels to induce signaling events that promote Golgi fragmentation and increase vesicular traffic into the pathogen-occupied vacuole that the bacterium parasitizes. As several intracellular microbial pathogens destabilize the Golgi to drive their infection cycles and changes in Golgi morphology is also linked to cancer and neurodegenerative disorder progression, this study identifies C1P as a potential broad-spectrum therapeutic target for infectious and non-infectious diseases.

Skewing cPLA2α activity toward oxoeicosanoid production promotes neutrophil N2 polarization, wound healing, and the response to sepsis.

Uncontrolled inflammation is linked to poor outcomes in sepsis and wound healing, both of which proceed through distinct inflammatory and resolution phases. Eicosanoids are a class of bioactive lipids that recruit neutrophils and other innate immune cells. The interaction of ceramide 1-phosphate (C1P) with the eicosanoid biosynthetic enzyme cytosolic phospholipase A2 (cPLA2) reduces the production of a subtype of eicosanoids called oxoeicosanoids. We investigated the effect of shifting the balance in eicosanoid biosynthesis on neutrophil polarization and function. Knockin mice expressing a cPLA2 mutant lacking the C1P binding site (cPLA2αKI/KI mice) showed enhanced and sustained neutrophil infiltration into wounds and the peritoneum during the inflammatory phase of wound healing and sepsis, respectively. The mice exhibited improved wound healing and reduced susceptibility to sepsis, which was associated with an increase in anti-inflammatory N2-type neutrophils demonstrating proresolution behaviors and a decrease in proinflammatory N1-type neutrophils. The N2 polarization of cPLA2αKI/KI neutrophils resulted from increased oxoeicosanoid biosynthesis and autocrine signaling through the oxoeicosanoid receptor OXER1 and partially depended on OXER1-dependent inhibition of the pentose phosphate pathway (PPP). Thus, C1P binding to cPLA2α suppresses neutrophil N2 polarization, thereby impairing wound healing and the response to sepsis.

Fluoxetine restrains allergic inflammation by targeting an FcɛRI-ATP positive feedback loop in mast cells.

There is a clinical need for new treatment options addressing allergic disease. Selective serotonin reuptake inhibitors (SSRIs) are a class of antidepressants that have anti-inflammatory properties. We tested the effects of the SSRI fluoxetine on IgE-induced function of mast cells, which are critical effectors of allergic inflammation. We showed that fluoxetine treatment of murine or human mast cells reduced IgE-mediated degranulation, cytokine production, and inflammatory lipid secretion, as well as signaling mediated by the mast cell activator ATP. In a mouse model of systemic anaphylaxis, fluoxetine reduced hypothermia and cytokine production. Fluoxetine was also effective in a model of allergic airway inflammation, where it reduced bronchial responsiveness and inflammation. These data show that fluoxetine suppresses mast cell activation by impeding an FcɛRI-ATP positive feedback loop and support the potential repurposing of this SSRI for use in allergic disease.

The Complete Genome Sequence of the Simalia boeleni, the Boelen's Python.

The Boelen's Python (Simalia boeleni) is a python endemic to the mountains of New Guinea. We present the whole genome sequence of this species. Illumina sequencing was performed on a genetic sample from a single individual. The reads were assembled using a de novo method followed by a series of references from related species for finishing. The raw and assembled data is publicly available via Genbank: Sequence Read Archive (SRR19167501) and assembled genome (JANKYG000000000).

Ceramide Kinase Inhibition Drives Ferroptosis and Sensitivity to Cisplatin in Mutant KRAS Lung Cancer by Dysregulating VDAC-Mediated Mitochondria Function.

Ceramide kinase (CERK) is the mammalian lipid kinase from which the bioactive sphingolipid, ceramide-1-phosphate (C1P), is derived. CERK has been implicated in several promalignant phenotypes with little known as to mechanistic underpinnings. In this study, the mechanism of how CERK inhibition decreases cell survival in mutant (Mut) KRAS non-small cell lung cancer (NSCLC), a major lung cancer subtype, was revealed. Specifically, NSCLC cells possessing a KRAS mutation were more responsive to inhibition, downregulation, and genetic ablation of CERK compared with those with wild-type (WT) KRAS regarding a reduction in cell survival. Inhibition of CERK induced ferroptosis in Mut KRAS NSCLC cells, which required elevating VDAC-regulated mitochondria membrane potential (MMP) and the generation of cellular reactive oxygen species (ROS). Importantly, through modulation of VDAC, CERK inhibition synergized with the first-line NSCLC treatment, cisplatin, in reducing cell survival and in vivo tumor growth. Further mechanistic studies indicated that CERK inhibition affected MMP and cell survival by limiting AKT activation and translocation to mitochondria, and thus, blocking VDAC phosphorylation and tubulin recruitment. IMPLICATIONS: Our findings depict how CERK inhibition may serve as a new key point in combination therapeutic strategy for NSCLC, specifically precision therapeutics targeting NSCLC possessing a KRAS mutation.

A rapid and adaptable lipidomics method for quantitative UPLC-mass spectrometric analysis of phosphatidylethanolamine and phosphatidylcholine in vitro, and in cells.

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are highly prevalent phospholipids in mammalian membranes. There are currently no methods for detection of minute levels of these phospholipids or simultaneously with products of the utilization of these phospholipid substrates by phospholipase A2 (PLA2) enzymes. To examine the substrate utilization of PE and PC by PLA2, we developed a method to accurately detect and measure specific forms of PE and PC as low as 50 femtomoles. Validation of this method consisted of an enzymatic assay to monitor docosahexaenoic acid and arachidonic acid release from the hydrolysis of PE and PC by group IV phospholipase A2 (cPLA2α) coupled to the generation of lyso-PE (LPE) and lyso-PC (LPC). In addition, the PE and PC profiles of RAW 264.7 macrophages were monitored with zymosan/lipopolysaccharide-treatment. Finally, genetic validation for the specificity of the method consisted of the downregulation of two biosynthetic enzymes responsible for the production of PE and PC, choline kinase A (CHKA) and ethanolamine kinase 1 (ETNK1). This new UPLC ESI-MS/MS method provides accurate and highly sensitive detection of PE and PC species containing AA and DHA allowing for the specific examination of the substrate utilization of these phospholipids by PLA2 in vitro and in cells.

Serine/Arginine-Rich Splicing Factor 3 Modulates the Alternative Splicing of Cytoplasmic Polyadenylation Element Binding Protein 2.

Triple negative breast cancer (TNBC) has an unusually low 5-year survival rate linked to higher metastatic rates. Our laboratory recently delineated a role for the alternative RNA splicing (AS) of cytoplasmic polyadenylation element binding protein 2 (CPEB2), via inclusion/exclusion of exon 4, in the metastasis of TNBC. In these studies, the mechanism governing the inclusion/exclusion of exon 4 was examined. Specifically, the RNA trans-factor, SRSF3, was found to be explicitly associated with CPEB2 exon 4. A SRSF3 consensus sequence was identified in exon 4, and mutation of this sequence abolished the association of SRSF3. The expression of SRSF3 was upregulated in TNBC cells upon the acquisition of anoikis resistance correlating with a reduction in the CPEB2A/B ratio. Importantly, downregulation of SRSF3 in these cells by siRNA induced the exclusion of exon 4 in cells increasing the ratio of CPEB2A (exon 4 excluded) to CPEB2B (exon 4 included). Downregulation of SRSF3 also reversed the CPEB2A/B ratio of a wild-type CPEB2 exon 4 minigene and endogenous CPEB2 pre-mRNA, but not a mutant CPEB2 minigene with the SRSF3 RNA cis-element ablated. SRSF3 downregulation ablated the anoikis resistance of TNBC cells, which was "rescued" by ectopic expression of CPEB2B. Finally, analysis of The Cancer Genome Atlas database showed a positive relationship between SRSF3 expression and lower CPEB2A/B ratios in aggressive breast cancers. IMPLICATIONS: These findings demonstrate that SRSF3 modulates CPEB2 AS to induce the expression of the CPEB2B isoform that drives TNBC phenotypes correlating with aggressive human breast cancer. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/17/9/1920/F1.large.jpg.

The interaction of ceramide 1-phosphate with group IVA cytosolic phospholipase A2 coordinates acute wound healing and repair.

The sphingolipid ceramide 1-phosphate (C1P) directly binds to and activates group IVA cytosolic phospholipase A2 (cPLA2α) to stimulate the production of eicosanoids. Because eicosanoids are important in wound healing, we examined the repair of skin wounds in knockout (KO) mice lacking cPLA2α and in knock-in (KI) mice in which endogenous cPLA2α was replaced with a mutant form having an ablated C1P interaction site. Wound closure rate was not affected in the KO or KI mice, but wound maturation was enhanced in the KI mice compared to that in wild-type controls. Wounds in KI mice displayed increased infiltration of dermal fibroblasts into the wound environment, increased wound tensile strength, and a higher ratio of type I:type III collagen. In vitro, primary dermal fibroblasts (pDFs) from KI mice showed substantially increased collagen deposition and migration velocity compared to pDFs from wild-type and KO mice. KI mice also showed an altered eicosanoid profile of reduced proinflammatory prostaglandins (PGE2 and TXB2) and an increased abundance of certain hydroxyeicosatetraenoic acid (HETE) species. Specifically, an increase in 5-HETE enhanced dermal fibroblast migration and collagen deposition. This gain-of-function role for the mutant cPLA2α was also linked to the relocalization of cPLA2α and 5-HETE biosynthetic enzymes to the cytoplasm and cytoplasmic vesicles. These findings demonstrate the regulation of key wound-healing mechanisms in vivo by a defined protein-lipid interaction and provide insights into the roles that cPLA2α and eicosanoids play in orchestrating wound repair.

Controlling Mast Cell Activation and Homeostasis: Work Influenced by Bill Paul That Continues Today.

Mast cells are tissue resident, innate immune cells with heterogenous phenotypes tuned by cytokines and other microenvironmental stimuli. Playing a protective role in parasitic, bacterial, and viral infections, mast cells are also known for their role in the pathogenesis of allergy, asthma, and autoimmune diseases. Here, we review factors controlling mast cell activation, with a focus on receptor signaling and potential therapies for allergic disease. Specifically, we will discuss our work with FcεRI and FγR signaling, IL-4, IL-10, and TGF-ß1 treatment, and Stat5. We conclude with potential therapeutics for allergic disease. Much of these efforts have been influenced by the work of Bill Paul. With many mechanistic targets for mast cell activation and different classes of therapeutics being studied, there is reason to be hopeful for continued clinical progress in this area.