Metallosphaera javensis sp. nov., a novel species of thermoacidophilic archaea, isolated from a volcanic area.

A novel thermoacidophilic archeaon, strain J1T (=DSM 112778T,=JCM 34702T), was isolated from a hot pool in a volcanic area of Java, Indonesia. Cells of the strain were irregular, motile cocci of 1.0-1.2 µm diameter. Aerobic, organoheterotrophic growth with casamino acids was observed at an optimum temperature of 70 °C in a range of 55-78 °C and at an optimum pH of 3 in a range of 1.5 to 5. Various organic compounds were utilized, including a greater variety of sugars than has been reported for growth of other species of the genus. Chemolithoautotrophic growth was observed with reduced sulphur compounds, including mineral sulphides. Ferric iron was reduced during anaerobic growth with elemental sulphur. Cellular lipids were calditoglycerocaldarchaeol and caldarchaeol with some derivates. The organism contained the respiratory quinone caldariellaquinone. On the basis of phylogenetic and chemotaxonomic comparison with its closest relatives, it was concluded that strain J1T represents a novel species, for which the name Metallosphaera javensis is proposed. Low DNA-DNA relatedness values (16S rRNA gene <98.4%, average nucleotide identity (ANI) <80.1%) distinguished J1T from other species of the genus Metallosphaera and the DNA G+C content of 47.3% is the highest among the known species of the genus.

Centralised pathology service for sentinel node biopsy in oral cavity cancer: The Southeast England Consortium experience.

BACKGROUND: Sentinel lymph node biopsy is an increasingly recognised option for accurate staging and subsequent management of the clinically negative neck in early stage oral cavity squamous cell carcinoma. However, the technique is currently underused due to several logistic constraints including increased burden on pathology services. Here, we describe the feasibility of an outsourced centralised pathology processing and reporting service for sentinel lymph node biopsies in oral cavity squamous cell carcinoma. PATIENTS AND METHODS: The Southeast England Consortium comprises four surgical centres utilising a central pathology service. Consecutive cases between January 2016 and February 2020 were retrospectively evaluated for survival outcomes and laboratory turnaround times. RESULTS: Twenty-eight per cent from a cohort of 139 patients had positive sentinel nodes. There was a trend towards greater overall, disease-free and disease-specific survival (OS, DFS and DSS, respectively) in sentinel node negative compared to sentinel node positive patients, but these differences were not statistically significant. The sensitivity, negative predictive value and false negative rate were 92.8%, 97.0% and 6.8%, respectively. The mean and mode laboratory TAT were 5 and 4 working days, respectively. CONCLUSION: An outsourced centralised pathology service is a feasible option to widen the availability of sentinel node biopsy in oral cavity squamous cell carcinoma.

Aspirin-triggered proresolving mediators stimulate resolution in cancer.

Inflammation in the tumor microenvironment is a strong promoter of tumor growth. Substantial epidemiologic evidence suggests that aspirin, which suppresses inflammation, reduces the risk of cancer. The mechanism by which aspirin inhibits cancer has remained unclear, and toxicity has limited its clinical use. Aspirin not only blocks the biosynthesis of prostaglandins, but also stimulates the endogenous production of anti-inflammatory and proresolving mediators termed aspirin-triggered specialized proresolving mediators (AT-SPMs), such as aspirin-triggered resolvins (AT-RvDs) and lipoxins (AT-LXs). Using genetic and pharmacologic manipulation of a proresolving receptor, we demonstrate that AT-RvDs mediate the antitumor activity of aspirin. Moreover, treatment of mice with AT-RvDs (e.g., AT-RvD1 and AT-RvD3) or AT-LXA4 inhibited primary tumor growth by enhancing macrophage phagocytosis of tumor cell debris and counter-regulating macrophage-secreted proinflammatory cytokines, including migration inhibitory factor, plasminogen activator inhibitor-1, and C-C motif chemokine ligand 2/monocyte chemoattractant protein 1. Thus, the pro-resolution activity of AT-resolvins and AT-lipoxins may explain some of aspirin's broad anticancer activity. These AT-SPMs are active at considerably lower concentrations than aspirin, and thus may provide a nontoxic approach to harnessing aspirin's anticancer activity.

Evaluation of appropriate vancomycin prescribing for the prevention of newborn group B streptococcal infections in a community hospital obstetrics service.

OBJECTIVES: The 2019 American College of Obstetricians and Gynecologists (ACOG) guidelines update for the prevention of perinatal group B Streptococcus (GBS) infections stipulate that vancomycin should be reserved to treat penicillin-allergic women at high risk for anaphylaxis with documented GBS resistance to clindamycin. Protocols and policies were adapted at the community hospital to incorporate these new guidelines. The primary objective of this research was to evaluate institutional compliance to these guidelines and secondarily, clinical outcomes. METHODS: Clinical pharmacists, in collaboration with an obstetrician, performed this hospital-based study. All instances of intravenous (IV) vancomycin therapy in GBS-positive patients were assessed from 1/1/2018 through 1/1/2021 and compared to the 2010 and 2019 ACOG guidelines. Treatment was analyzed to determine the appropriateness of both indication for use and dosage regimen as co-primary endpoints. Secondary endpoints included renal monitoring parameters, suspected adverse reactions, and early onset GBS disease in newborns, specifically sepsis, meningitis, and/or pneumonia. RESULTS: L&D admissions during the study period included 15,129 patients. All 30 L&D patients who received IV vancomycin for GBS prophylaxis were included in the study. This project demonstrated low compliance to the ACOG guidelines and identified previously unrecognized opportunities for improvement. CONCLUSIONS: The low compliance observed in this study, with the exception of documenting GBS status, occurred in spite of hospital adoption of a GBS order set, an updated vancomycin protocol and targeted education of clinical pharmacists. Assessment of the causes of noncompliance identified several potential corrective actions, especially in ordering and monitoring vancomycin.

Resolvin D4 attenuates the severity of pathological thrombosis in mice.

Deep vein thrombosis (DVT) is a common cardiovascular disease with a major effect on quality of life, and safe and effective therapeutic measures to efficiently reduce existent thrombus burden are scarce. Using a comprehensive targeted liquid chromatography-tandem mass spectrometry-based metabololipidomics approach, we established temporal clusters of endogenously biosynthesized specialized proresolving mediators (SPMs) and proinflammatory and prothrombotic lipid mediators during DVT progression in mice. Administration of resolvin D4 (RvD4), an SPM that was enriched at the natural onset of thrombus resolution, significantly reduced thrombus burden, with significantly less neutrophil infiltration and more proresolving monocytes in the thrombus, as well as an increased number of cells in an early apoptosis state. Moreover, RvD4 promoted the biosynthesis of other D-series resolvins involved in facilitating resolution of inflammation. Neutrophils from RvD4-treated mice were less susceptible to an ionomycin-induced release of neutrophil extracellular traps (NETs), a meshwork of decondensed chromatin lined with histones and neutrophil proteins critical for DVT development. These results suggest that delivery of SPMs, specifically RvD4, modulates the severity of thrombo-inflammatory disease in vivo and improves thrombus resolution.

Resolution of sickle cell disease-associated inflammation and tissue damage with 17R-resolvin D1.

Resolvins (Rvs), endogenous lipid mediators, play a key role in the resolution of inflammation. Sickle cell disease (SCD), a genetic disorder of hemoglobin, is characterized by inflammatory and vaso-occlusive pathologies. We document altered proresolving events following hypoxia/reperfusion in humanized SCD mice. We demonstrate novel protective actions of 17R-resolvin D1 (17R-RvD1; 7S, 8R, 17R-trihydroxy-4Z, 9E, 11E, 13Z, 15E, 19Z-docosahexaenoic acid) in reducing ex vivo human SCD blood leukocyte recruitment by microvascular endothelial cells and in vivo neutrophil adhesion and transmigration. In SCD mice exposed to hypoxia/reoxygenation, oral administration of 17R -RvD1 reduces systemic/local inflammation and vascular dysfunction in lung and kidney. The mechanism of action of 17R-RvD1 involves (1) enhancement of SCD erythrocytes and polymorphonuclear leukocyte efferocytosis, (2) blunting of NF-κB activation, and (3) a reduction in inflammatory cytokines, vascular activation markers, and E-selectin expression. Thus, 17R-RvD1 might represent a new therapeutic strategy for the inflammatory vasculopathy of SCD.

Perineural and lymphovascular invasion in squamous cell carcinoma of the tongue.

BACKGROUND: Perineural invasion (PNI) and lymphovascular invasion (LVI) may be adverse prognostic indicators in squamous cell carcinoma (SCC) of the tongue. METHODS: The percentages of histological PNI and LVI were determined in 335 patients with tongue SCC. Sixty tumours originally reported as negative for these features were tested to determine how many more were positive with "immunohistochemical enhancement." RESULTS: PNI was found in 141 (42.1%) and LVI in 51 (15.2%) patients. 79.4% of the 141 patients who had PNI and 72.6% of the 51 with LVI had a T3 or T4 tumour. Lymph node metastasis was identified in 145 (51.2%) of the 280 patients who had undergone neck dissection; 58.2% of the 141 patients with PNI and 80.4% of the 51 patients with LVI had lymph node metastasis. There was a highly statistically significant correlation between PNI with increasing pT (P < .00001) and pN (P < .0001) stage, and a statistically significant correlation between LVI and pT stage (P < .001), the association of LVI with pN status could not be reliably tested statistically. Immunohistochemistry for S100 identified five further cases of PNI, but review of the original H&E showed the feature was present in four and had been missed at original reporting. CD31 identified three further possible cases of LVI and D2-40 none. The endothelium of some vascular channels was positive for both CD31 and D2-40 and cross-reactivity with other cells compromised interpretation. CONCLUSIONS: Histological identification of PNI and LVI per se remains of uncertain prognostic significance. "Immunohistochemical enhancement" offered little benefit.

Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132.

Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling molecules with cardioprotective and vasodilatory actions. We recently showed that 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via G protein-coupled receptors, with evidence supporting the existence of a specific high-affinity receptor. Identification of a hematopoietic-specific EET receptor would enable genetic interrogation of EET signaling pathways, and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify an EET receptor based on the expression of G protein-coupled receptors in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are expressed in three EET-responsive cell lines, but not expressed in an EET-unresponsive line. Of these, only recombinant GPR132 showed EET-responsiveness in vitro, using a luminescence-based ß-arrestin recruitment assay. Knockdown of zebrafish gpr132b prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to high-affinity EET receptors, GPR132 is reported to respond to additional hydroxy-fatty acids in vitro, and we found that these same hydroxy-fatty acids enhance hematopoiesis in the zebrafish. We conducted structure-activity relationship analyses using both cell culture and zebrafish assays on diverse medium-chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, whereas unoxygenated or saturated fatty acids had lower activity. Absence of the carbon-1 position carboxylic acid prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of oxygenated polyunsaturated fatty acids to enhance both embryonic and adult hematopoiesis.

Cysteinyl maresins regulate the prophlogistic lung actions of cysteinyl leukotrienes.

BACKGROUND: Cysteinyl leukotrienes (CysLTs) are potent prophlogistic mediators in asthmatic patients; however, inhibition of CysLT receptor 1 is not a consistently effective treatment, suggesting additional regulatory mechanisms. Other cysteinyl-containing lipid mediators (LMs) derived from docosahexaenoic acid, namely maresin conjugates in tissue regeneration (MCTRs), were recently discovered. Therefore their production and actions in the lung are of considerable interest. OBJECTIVE: We sought to determine MCTR production, bioactions, and mechanisms in the human lung and in patients with experimental allergic airway inflammation. METHODS: LM metabololipidomic profiling of the lung was performed by using liquid chromatography with tandem mass spectrometry. Donor-derived human precision-cut lung slices were exposed to leukotriene (LT) D4, MCTRs, or both before determination of airway contraction. The actions of exogenous MCTRs on murine allergic host responses were determined in the setting of ovalbumin- and house dust mite-induced lung inflammation. RESULTS: Lipidomic profiling showed that the most abundant cysteinyl LMs in healthy human lungs were MCTRs, whereas CysLTs were most prevalent in patients with disease. MCTRs blocked LTD4-initiated airway contraction in human precision-cut lung slices. In mouse allergic lung inflammation MCTRs were present with temporally regulated production. With ovalbumin-induced inflammation, MCTR1 was most potent for promoting resolution of eosinophils, and MCTR3 potently decreased airway hyperreactivity to methacholine, bronchoalveolar lavage fluid albumin, and serum IgE levels. MCTR1 and MCTR3 inhibited lung eosinophilia after house dust mite-induced inflammation. CONCLUSION: These results identified lung MCTRs that blocked human LTD4-induced airway contraction and promoted resolution of murine allergic airway responses when added exogenously. Together, these findings uncover proresolving mechanisms for lung responses that can be disrupted in patients with disease.

Endogenous Specialized Proresolving Mediator Profiles in a Novel Experimental Model of Lymphatic Obstruction and Intestinal Inflammation in African Green Monkeys.

Changes in the intestinal lymphatic vascular system, such as lymphatic obstruction, are characteristic features of inflammatory bowel diseases. The lymphatic vasculature forms a conduit to enable resolution of inflammation; this process is driven by specialized endogenous proresolving mediators (SPMs). To evaluate contributions of lymphatic obstruction to intestinal inflammation and to study profiles of SPMs, we generated a novel animal model of lymphatic obstruction using African green monkeys. Follow-up studies were performed at 7, 21, and 61 days. Inflammation was determined by histology. Luminex assays were performed to evaluate chemokine and cytokine levels. In addition, lipid mediator metabololipidomic profiling was performed to identify SPMs. After 7 days, lymphatic obstruction resulted in a localized inflammatory state, paralleled by an increase in inflammatory chemokines and cytokines, which were found to be up-regulated after 7 days but returned to baseline after 21 and 61 days. At the same time, a distinct pattern of SPMs was profiled, with an increase for D-series resolvins, protectins, maresins, and lipoxins at 61 days. These results indicate that intestinal lymphatic obstruction can lead to an acute inflammatory state, accompanied by an increase in proinflammatory mediators, followed by a phase of resolution, paralleled by an increase and decrease of respective SPMs.

Specialized pro-resolving lipid mediators are differentially altered in peripheral blood of patients with multiple sclerosis and attenuate monocyte and blood-brain barrier dysfunction.

Chronic inflammation is a key pathological hallmark of multiple sclerosis (MS) and suggests that resolution of inflammation, orchestrated by specialized pro-resolving lipid mediators (LM), is impaired. Here, through targeted-metabololipidomics in peripheral blood of patients with MS, we revealed that each disease form was associated with distinct LM profiles that significantly correlated with disease severity. In particular, relapsing and progressive MS patients were associated with high eicosanoids levels, whereas the majority of pro-resolving LM were significantly reduced or below limits of detection and correlated with disease progression. Furthermore, we found impaired expression of several pro-resolving LM biosynthetic enzymes and receptors in blood-derived leukocytes of MS patients. Mechanistically, differentially expressed mediators like LXA4, LXB4, RvD1 and PD1 reduced MS-derived monocyte activation and cytokine production, and inhibited inflammation-induced blood-brain barrier dysfunction and monocyte transendothelial migration. Altogether, these findings reveal peripheral defects in the resolution pathway in MS, suggesting pro-resolving LM as novel diagnostic biomarkers and potentially safe therapeutics.

Biosynthetic metabolomes of cysteinyl-containing immunoresolvents.

Resolution of inflammation is an active process regulated by specialized proresolving mediators where we identified 3 new pathways producing allylic epoxide-derived mediators that stimulate regeneration [i.e., peptido-conjugates in tissue regeneration (CTRs)]. Here, using self-limited Escherichia coli peritonitis in mice, we identified endogenous maresin (MaR) CTR (MCTR), protectin (PD) CTR (PCTR), and resolvin CTR in infectious peritoneal exudates and distal spleens, as well as investigated enzymes involved in their biosynthesis. PCTRs were identified to be temporally regulated in peritoneal exudates and spleens. PCTR1 and MCTR1 were each produced by human recombinant leukotriene (LT) C4 synthase (LTC4S) and glutathione S-transferases (GSTs) [microsomal GST (mGST)2, mGST3, and GST-µ (GSTM)4] from their epoxide precursors [16S,17S-epoxy-PD (ePD) and 13S,14S-epoxy-MaR (eMaR)], with preference for GSTM4. Both eMaR and ePD inhibited LTB4 production by LTA4 hydrolase. LTC4S, mGST2, mGST3, and GSTM4 were each expressed in human M1- and M2-like macrophages where LTC4S inhibition increased CTRs. Finally, PCTR1 showed potent analgesic action. These results demonstrate CTR biosynthesis in mouse peritonitis, human spleens, and human macrophages, as well as identification of key enzymes in these pathways. Moreover, targeting LTC4S increases CTR metabolomes, giving a new strategy to stimulate resolution and tissue regeneration.-Jouvene, C. C., Shay, A. E., Soens, M. A., Norris, P. C., Haeggström, J. Z., Serhan, C. N. Biosynthetic metabolomes of cysteinyl-containing immunoresolvents.

Salt-tolerant Acidihalobacter and Acidithiobacillus species from Vulcano (Italy) and Milos (Greece).

Ferrous iron- and sulfur-oxidizing Acidihalobacter species and similar so far unclassified bacteria have been isolated from the islands of Vulcano (Italy) and Milos (Greece), specifically from where seawater was acidified at sulfide-rich geothermal sites. Acidithiobacillus species which tolerated concentrations of chloride that inhibit most Acidithiobacillus spp. were also isolated from sites on both islands: these were At. thiooxidans strains and an unclassified species, Acidithiobacillus sp. strain V1. The potential of salt-tolerant acidophiles for industrial application in promoting copper extraction from mineral sulfides where chloride is naturally present at concentrations which would inhibit most acidophiles, or where seawater rather than fresh water is available, appears to be limited by the sensitivity of ferrous-iron oxidizing Acidihalobacter spp. to copper. However, tolerance of copper and chloride shown by At. thiooxidans strain A7 suggests it could oxidize sulfur and benefit acid leaching if ferric iron or copper was provided as the primary oxidant of sulfide ores.

Acidithiobacillus ferrianus sp. nov.: an ancestral extremely acidophilic and facultatively anaerobic chemolithoautotroph.

Strain MG, isolated from an acidic pond sediment on the island of Milos (Greece), is proposed as a novel species of ferrous iron- and sulfur-oxidizing Acidithiobacillus. Currently, four of the eight validated species of this genus oxidize ferrous iron, and strain MG shares many key characteristics with these four, including the capacities for catalyzing the oxidative dissolution of pyrite and for anaerobic growth via ferric iron respiration. Strain MG also grows aerobically on hydrogen and anaerobically on hydrogen coupled to ferric iron reduction. While the 16S rRNA genes of the iron-oxidizing Acidi-thiobacillus species (and strain MG) are located in a distinct phylogenetic clade and are closely related (98-99% 16S rRNA gene identity), genomic relatedness indexes (ANI/dDDH) revealed strong genomic divergence between strain MG and all sequenced type strains of the taxon, and placed MG as the first cultured representative of an ancestral phylotype of iron oxidizing acidithiobacilli. Strain MG is proposed as a novel species, Acidithiobacillus ferrianus sp. nov. The type strain is MGT (= DSM 107098T = JCM 33084T). Similar strains have been found as isolates or indicated by cloned 16S rRNA genes from several mineral sulfide mine sites.

15-epi-Lipoxin A4, Resolvin D2, and Resolvin D3 Induce NF-κB Regulators in Bacterial Pneumonia.

Specialized proresolving mediators (SPMs) decrease NF-κB activity to prevent excessive tissue damage and promote the resolution of acute inflammation. Mechanisms for NF-κB regulation by SPMs remain to be determined. In this study, after LPS challenge, the SPMs 15-epi-lipoxin A4 (15-epi-LXA4), resolvin D1, resolvin D2, resolvin D3, and 17-epi-resolvin D1 were produced in vivo in murine lungs. In LPS-activated human bronchial epithelial cells, select SPMs increased expression of the NF-κB regulators A20 and single Ig IL-1R-related molecule (SIGIRR). Of interest, 15-epi-LXA4 induced A20 and SIGIRR in an lipoxin A4 receptor/formyl peptide receptor 2 (ALX/FPR2) receptor-dependent manner in epithelial cells and in murine pneumonia. This SPM regulated NF-κB-induced cytokines to decrease pathogen-mediated inflammation. In addition to dampening lung inflammation, surprisingly, 15-epi-LXA4 also enhanced pathogen clearance with increased antimicrobial peptide expression. Taken together, to our knowledge these results are the first to identify endogenous agonists for A20 and SIGIRR expression to regulate NF-κB activity and to establish mechanisms for NF-κB regulation by SPMs for pneumonia resolution.

Identification and Complete Stereochemical Assignments of the New Resolvin Conjugates in Tissue Regeneration in Human Tissues that Stimulate Proresolving Phagocyte Functions and Tissue Regeneration.

Resolvin conjugates in tissue regeneration (RCTRs) are new chemical signals that accelerate resolution of inflammation, infection, and tissue regeneration. Herein, using liquid chromatography-tandem mass spectrometry-based metabololipidomics, we identified RCTRs in human spleen, lymph node, bone marrow, and brain. In human spleen incubated with Staphylococcus aureus, endogenous RCTRs were increased along with conversion of deuterium-labeled docosahexaenoic acid, conferring pathway activation. Physical and biological properties of endogenous RCTRs were matched with those prepared by total organic synthesis. The complete stereochemical assignment of bioactive RCTR1 is 8R-glutathionyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, RCTR2 is 8R-cysteinylglycinyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, and RCTR3 is 8R-cysteinyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid. These stereochemically defined RCTRs stimulated human macrophage phagocytosis, efferocytosis, and planaria tissue generation. Proteome profiling demonstrated that RCTRs regulated both proinflammatory and anti-inflammatory cytokines with human macrophages. In microfluidic chambers, the three RCTRs limited human polymorphonuclear cell migration. In hind-limb ischemia-reperfusion-initiated organ injury, both RCTR2 and RCTR3 reduced polymorphonuclear cell infiltration into lungs. In infectious peritonitis, RCTR1 shortened the resolution intervals. Each RCTR (1 nmol/L) accelerated planaria tissue regeneration by approximately 0.5 days, with direct comparison to both maresin and protectin CTRs. Together, these results identify a new bioactive RCTR (ie, RCTR3) in human tissues and establish the complete stereochemistry and rank-order potencies of three RCTRs in vivo. Moreover, RCTR1, RCTR2, and RCTR3 each exert potent anti-inflammatory and proresolving actions with human leukocytes.

Metabololipidomic profiling of functional immunoresolvent clusters and eicosanoids in mammalian tissues.

Metabolomics enables a systems approach to interrogate the bioactive mediators, their pathways and further metabolites involved in the physiology and pathophysiology of human and animal tissues. New metabololipidomic approaches with mass spectrometry presented in this brief review can now be utilized for the identification and profiling of lipid mediator networks that control inflammation-resolution in human blood and healthy and diseased solid tissues. Coagulation of blood is a protective response that prevents excessive bleeding on injury of blood vessels. Here, we review novel approaches to understand the relationship(s) between coagulation and resolution of inflammation and infection. To determine whether coagulation is involved in host-protective actions by lipid mediators, we used a metabololipidomic-based profiling approach with human whole blood (WB) during coagulation. We identified recently temporal clusters of endogenously produced pro-thrombotic and proinflammatory lipid mediators (eicosanoids), as well as specialized proresolving mediators (SPMs) in this vital process. In addition to the classic eicosanoids (prostaglandins, thromboxanes and leukotrienes), a specific SPM cluster was identified that consists of resolvin E1 (RvE1), RvD1, RvD5, lipoxin B4, and maresin 1, each of which present at bioactive concentrations (0.1-1 nM). The removal of adenosine from coagulating blood samples significantly enhances SPM amounts and unleashes the biosynthesis of RvD3, RvD4, and RvD6 evident following rapid snap freezing with centrifugation before extraction and LC-MS-MS. The classic cyclooxygenase inhibitors, celecoxib and indomethacin, that block thromboxanes and prostanoids do not block production of the clot-driven SPM cluster. Unbiased mass cytometry analysis demonstrated that the SPM cluster produced in human blood targets leukocytes at the single-cell level, directly activating extracellular signaling in human neutrophils and monocytes. Human whole blood treated with the components of this SPM cluster enhanced both phagocytosis and killing of Escherichia coli by leukocytes. Thus, we identified a pro-resolving lipid mediator circuit and specific SPM cluster that promotes host defense. This new lipid mediator (LM)-SPM metabololipidomic approach now provides accessible metabolomic profiles in healthy and diseased human tissues, including cancer, for precision and personalized medicine.

Cytochromes in anaerobic growth of Acidithiobacillus ferrooxidans.

The mineral sulfide-oxidising Acidithiobacillus ferrooxidans has been extensively studied over many years but some fundamental aspects of its metabolism remain uncertain, particularly with regard to its anaerobic oxidation of sulfur. This label-free, liquid chromatography-electron spray ionisation-mass spectrometry-based proteomic analysis estimated relative protein abundance during aerobic and anaerobic growth of At. ferrooxidans. One of its two bc1 complexes, that encoded by the petII operon, was strongly implicated in anaerobic ferric iron-coupled sulfur oxidation, probably in conjunction with two cytochromes. These two cytochromes are homologs of the Cyc2 and Cyc1 proteins that are involved in ferrous iron oxidation. The previously undetected cytochromes apparently associated with anaerobic growth in At. ferrooxidans appear to be absent in many other ferrous iron-oxidising acidophiles that can also reduce ferric iron, which suggests a diversity in the ferric-iron-coupled sulfur oxidation pathways. For aerobic growth of At. ferrooxidans, this analysis was consistent with the generally accepted mechanism for its oxidation of ferrous iron. Unexpectedly, proteins encoded by the petI operon were not abundant and generally not detected in the proteomic analyses of cells grown aerobically on sulfur, although there was some expression of genes of the petI and petII operons in these cells.

NLRP3 Inflammasome Deficiency Protects against Microbial Sepsis via Increased Lipoxin B4 Synthesis.

RATIONALE: Sepsis, a life-threatening organ dysfunction caused by a dysregulated host response to infection, is a major public health concern with high mortality and morbidity. Although inflammatory responses triggered by infection are crucial for host defense against invading microbes, the excessive inflammation often causes tissue damage leading to organ dysfunction. Resolution of inflammation, an active immune process mediated by endogenous lipid mediators (LMs), is important to maintain host homeostasis. OBJECTIVES: We sought to determine the role of the nucleotide-binding domain, leucine-rich repeat-containing receptor, pyrin domain-containing-3 (NLRP3) inflammasome in polymicrobial sepsis and regulation of LM biosynthesis. METHODS: We performed cecal ligation and puncture (CLP) using mice lacking NLRP3 inflammasome-associated molecules to assess mortality. Inflammation was evaluated by using biologic fluids including plasma, bronchoalveolar, and peritoneal lavage fluid. Local acting LMs in peritoneal lavage fluid from polymicrobacterial septic mice were assessed by mass spectrometry-based metabololipidomics. MEASUREMENTS AND MAIN RESULTS: Genetic deficiency of NLRP3 inhibited inflammatory responses and enhanced survival of CLP-induced septic mice. NLRP3 deficiency reduced proinflammatory LMs and increased proresolving LM, lipoxin B4 (LXB4) in septic mice, and in macrophages stimulated with LPS and ATP. Activation of the NLRP3 inflammasome induced caspase-7 cleavage and pyroptosis. Caspase-7 deficiency similarly reduced inflammation and mortality in CLP-induced sepsis, and increased LXB4 production in vivo and in vitro. Exogenous application of LXB4 reduced inflammation, pyroptosis, and mortality of mice after CLP. CONCLUSIONS: Genetic deficiency of NLRP3 promoted resolution of inflammation in polymicrobial sepsis by relieving caspase-7-dependent repression of LXB4 biosynthesis, and increased survival potentially via LXB4 production and inhibition of proinflammatory cytokines.

Phospholipase A2 regulates eicosanoid class switching during inflammasome activation.

Initiation and resolution of inflammation are considered to be tightly connected processes. Lipoxins (LX) are proresolution lipid mediators that inhibit phlogistic neutrophil recruitment and promote wound-healing macrophage recruitment in humans via potent and specific signaling through the LXA4 receptor (ALX). One model of lipoxin biosynthesis involves sequential metabolism of arachidonic acid by two cell types expressing a combined transcellular metabolon. It is currently unclear how lipoxins are efficiently formed from precursors or if they are directly generated after receptor-mediated inflammatory commitment. Here, we provide evidence for a pathway by which lipoxins are generated in macrophages as a consequence of sequential activation of toll-like receptor 4 (TLR4), a receptor for endotoxin, and P2X7, a purinergic receptor for extracellular ATP. Initial activation of TLR4 results in accumulation of the cyclooxygenase-2-derived lipoxin precursor 15-hydroxyeicosatetraenoic acid (15-HETE) in esterified form within membrane phospholipids, which can be enhanced by aspirin (ASA) treatment. Subsequent activation of P2X7 results in efficient hydrolysis of 15-HETE from membrane phospholipids by group IVA cytosolic phospholipase A2, and its conversion to bioactive lipoxins by 5-lipoxygenase. Our results demonstrate how a single immune cell can store a proresolving lipid precursor and then release it for bioactive maturation and secretion, conceptually similar to the production and inflammasome-dependent maturation of the proinflammatory IL-1 family cytokines. These findings provide evidence for receptor-specific and combinatorial control of pro- and anti-inflammatory eicosanoid biosynthesis, and potential avenues to modulate inflammatory indices without inhibiting downstream eicosanoid pathways.