Loss of selenoprotein W in murine macrophages alters the hierarchy of selenoprotein expression, redox tone, and mitochondrial functions during inflammation.

Macrophages play a pivotal role in mediating inflammation and subsequent resolution of inflammation. The availability of selenium as a micronutrient and the subsequent biosynthesis of selenoproteins, containing the 21st amino acid selenocysteine (Sec), are important for the physiological functions of macrophages. Selenoproteins regulate the redox tone in macrophages during inflammation, the early onset of which involves oxidative burst of reactive oxygen and nitrogen species. SELENOW is a highly expressed selenoprotein in bone marrow-derived macrophages (BMDMs). Beyond its described general role as a thiol and peroxide reductase and as an interacting partner for 14-3-3 proteins, its cellular functions, particularly in macrophages, remain largely unknown. In this study, we utilized Selenow knock-out (KO) murine bone marrow-derived macrophages (BMDMs) to address the role of SELENOW in inflammation following stimulation with bacterial endotoxin lipopolysaccharide (LPS). RNAseq-based temporal analyses of expression of selenoproteins and the Sec incorporation machinery genes suggested no major differences in the selenium utilization pathway in the Selenow KO BMDMs compared to their wild-type counterparts. However, selective enrichment of oxidative stress-related selenoproteins and increased ROS in Selenow-/- BMDMs indicated anomalies in redox homeostasis associated with hierarchical expression of selenoproteins. Selenow-/- BMDMs also exhibited reduced expression of arginase-1, a key enzyme associated with anti-inflammatory (M2) phenotype necessary to resolve inflammation, along with a significant decrease in efferocytosis of neutrophils that triggers pathways of resolution. Parallel targeted metabolomics analysis also confirmed an impairment in arginine metabolism in Selenow-/- BMDMs. Furthermore, Selenow-/- BMDMs lacked the ability to enhance characteristic glycolytic metabolism during inflammation. Instead, these macrophages atypically relied on oxidative phosphorylation for energy production when glucose was used as an energy source. These findings suggest that SELENOW expression in macrophages may have important implications on cellular redox processes and bioenergetics during inflammation and its resolution.

The role of selenoproteins in neutrophils during inflammation.

Polymorphonuclear neutrophils (PMNs)-derived ROS are involved in the regulation of multiple functions of PMNs critical in both inflammation and its timely resolution. Selenium is an essential trace element that functions as a gatekeeper of cellular redox homeostasis in the form of selenoproteins. Despite their well-studied involvement in regulating functions of various immune cells, limited studies have focused on the regulation of selenoproteins in PMN and their associated functions. Ex-vivo treatment of murine primary bone marrow derived PMNs with bacterial endotoxin lipopolysaccharide (LPS) indicated temporal regulation of several selenoprotein genes at the mRNA level. However, only glutathione peroxidase 4 (Gpx4) was significantly upregulated, while Selenof, Selenow, and Gpx1 were significantly downregulated in a temporal manner at the protein level. Exposure of PMNs isolated from tRNASec (Trsp)fl/fl S100A8Cre (TrspN) PMN-specific selenoprotein knockout mice, to the Gram-negative bacterium, Citrobacter rodentium, showed decreased bacterial growth, reduced phagocytosis, as well as impaired neutrophil extracellular trap (NET) formation ability, when compared to the wild-type PMNs. Increased extracellular ROS production upon LPS stimulation was also observed in TrspN PMNs that was associated with upregulation of Alox12, Cox2, and iNOS, as well as proinflammatory cytokines such as TNFα and IL-1ß. Our data indicate that the inhibition of selenoproteome expression results in alteration of PMN proinflammatory functions, suggesting a potential role of selenoproteins in the continuum of inflammation and resolution.

Urine DNA biomarkers for hepatocellular carcinoma screening.

BACKGROUND: Hepatocellular carcinoma (HCC) occurs in a well-defined high-risk patient population, but better screening tests are needed to improve sensitivity and efficacy. Therefore, we investigated the use of urine circulating tumour DNA (ctDNA) as a screening test. METHODS: Candidate markers in urine were selected from HCC and controls. We then enrolled 609 patients from five medical centres to test the selected urine panel. A two-stage model was developed to combine AFP and urine panel as a screening test. RESULTS: Mutated TP53, and methylated RASSF1a, and GSTP1 were selected as the urine panel markers. Serum AFP outperformed the urine panel among all cases of HCC, but the urine panel identified 49% of HCC cases with low AFP < 20 ng/ml. Using the two-stage model, the combined AFP and urine panel identified 148 of the 186 HCC cases (79.6% sensitivity at 90% specificity), which was 30% more than the cases detected with serum AFP alone. It also increased early-stage HCC detection from 62% to 92% (BCLC stage 0), and 40% to 77% (BCLC stage A). CONCLUSION: Urine ctDNA has promising diagnostic utility in patients in HCC, especially in those with low AFP and can be used as a potential non-invasive HCC screening test.

Selenium-dependent metabolic reprogramming during inflammation and resolution.

Trace element selenium (Se) is incorporated as the 21st amino acid, selenocysteine, into selenoproteins through tRNA[Ser]Sec. Selenoproteins act as gatekeepers of redox homeostasis and modulate immune function to effect anti-inflammation and resolution. However, mechanistic underpinnings involving metabolic reprogramming during inflammation and resolution remain poorly understood. Bacterial endotoxin lipopolysaccharide (LPS) activation of murine bone marrow-derived macrophages cultured in the presence or absence of Se (as selenite) was used to examine temporal changes in the proteome and metabolome by multiplexed tandem mass tag-quantitative proteomics, metabolomics, and machine-learning approaches. Kinetic deltagram and clustering analysis indicated that addition of Se led to extensive reprogramming of cellular metabolism upon stimulation with LPS enhancing the pentose phosphate pathway, tricarboxylic acid cycle, and oxidative phosphorylation, to aid in the phenotypic transition toward alternatively activated macrophages, synonymous with resolution of inflammation. Remodeling of metabolic pathways and consequent metabolic adaptation toward proresolving phenotypes began with Se treatment at 0 h and became most prominent around 8 h after LPS stimulation that included succinate dehydrogenase complex, pyruvate kinase, and sedoheptulokinase. Se-dependent modulation of these pathways predisposed bone marrow-derived macrophages to preferentially increase oxidative phosphorylation to efficiently regulate inflammation and its timely resolution. The use of macrophages lacking selenoproteins indicated that all three metabolic nodes were sensitive to selenoproteome expression. Furthermore, inhibition of succinate dehydrogenase complex with dimethylmalonate affected the proresolving effects of Se by increasing the resolution interval in a murine peritonitis model. In summary, our studies provide novel insights into the role of cellular Se via metabolic reprograming to facilitate anti-inflammation and proresolution.

Crth2 receptor signaling down-regulates lipopolysaccharide-induced NF-κB activation in murine macrophages via changes in intracellular calcium.

Prostaglandin D2 and its cyclopentenone metabolites [cyclopentenone prostaglandins (CyPGs)], Δ12prostaglandin J2 and 15-deoxy-Δ12,14-prostaglandin J2, act through 2 GPCRs, d-type prostanoid 1 and the chemoattractant receptor homologous molecule expressed on type 2 T-helper cells (Crth2). In addition to its role in allergy and asthma, the role of Crth2 in the resolution of inflammation, to mediate the proresolving functions of endogenous CyPGs, is not well understood. We investigated the regulation of LPS or zymosan-induced inflammatory response by signals from the Crth2 receptor in macrophages that lack Crth2 expression [knockout (KO)]. Increased expression of proinflammatory genes, including Tnf-α, was observed in Crth2 KO cells. Targeting the endogenous biosynthetic pathway of CyPGs with indomethacin or HQL79, which inhibit cyclooxygenases or hematopoietic prostaglandin D synthase, respectively, or use of Crth2 antagonists recapitulated the proinflammatory phenotype as in Crth2 KO cells. Ligand-dependent activation of Crth2 by 13,14-dihydro-15-keto-prostaglandin D2 increased Ca2+ influx through store-operated Ca2+ entry (SOCE) accompanied by the up-regulation of stromal interaction molecule 1 and calcium release-activated calcium modulator 1 expression, suggesting that the proresolution effects of CyPG-dependent activation of SOCE could be mediated by Crth2 during inflammation. Interestingly, Crth2 signaling down-regulated the Ca2+-regulated heat stable protein 1 that stabilizes Tnf-α mRNA via the increased expression of microRNA 155 to dampen inflammatory responses triggered through the TNF-α-NF-κB axis. In summary, these studies present a novel regulatory role for Crth2 during inflammatory response in macrophages.-Diwakar, B. T., Yoast, R., Nettleford, S., Qian, F., Lee, T.-J., Berry, S., Huffnagle, I., Rossi, R. M., Trebak, M., Paulson, R. F., Prabhu, K. S. Crth2 receptor signaling down-regulates lipopolysaccharide-induced NF-κB activation in murine macrophages via changes in intracellular calcium.

Characterization of the hepatitis B virus DNA detected in urine of chronic hepatitis B patients.

BACKGROUND: Detection of human hepatitis B virus (HBV) DNA in the urine of patients with chronic hepatitis B infection (CHB) has been reported previously, suggesting urine could provide a potential route of horizontal HBV transmission. However, it is not clear whether the HBV DNA detected in urine is indeed full-length, infectious viral DNA. The aim of this study is to assess the potential infectivity of urine from patients with CHB and to correlate HBV DNA detection in urine with clinical parameters, such as serum viral load and HBeAg status. METHODS: Urine from 60 CHB patients with serum viral loads ranging from undetectable to 108 IU/mL were analyzed for HBV DNA and serum immune markers. HBV DNA was detected from total urine DNA and size-fractionated urine DNA (separated into ≤1 kb and > 1 kb fractions) by PCR analysis of six regions of the HBV genome. RESULTS: Twenty-seven of 59 (45.7%) patients with HBV serum viral load (≥20 IU/mL) contained at least 20 copies per mL of fragmented HBV DNA in urine detected in at least 1 of the 6 PCR assay regions. Only one patient contained HBV DNA detected by all six regions, and was found to have evidence of blood in the urine. Sixteen of 25 urine samples with high viral load (> 105 IU/mL) and 11 of 34 urine samples with low viral load (< 105 IU/mL) contained detectable HBV DNA. Twelve of 27 (44.44%) patients with detectable HBV DNA in urine were HBeAg positive, and only 5 of these HBeAg positive patients were in the group of 33 (15.15%) patients with no detectable HBV DNA in urine. By Fishers' exact test, HBV DNA in urine is significantly associated with high serum viral load (P = 0.0197) and HBeAg (P = 0.0203). CONCLUSIONS: We conclude that urine from CHB patients with healthy kidney function should not contain full-length HBV DNA, and therefore should not be infectious.

Self-reactivated mesostructured Ca-Al-O composite for enhanced high-temperature CO2 capture and carbonation/calcination cycles performance.

In this study, highly efficient high-temperature CO2 sorbents of calcium aluminate (Ca-Al-O) mesostructured composite were synthesized using presynthesized mesoporous alumina (MA) as a porous matrix to react with calcium nitrate through a microwave-assisted process. Upon annealing at 600 °C, a highly stable mesoporous structure composed of poorly crystalline Ca12Al14O33 phase and the CaO matrix was obtained. The Ca-Al-O mesostructured sorbents with a Ca(2+)/Al(3+) ratio of 5:1 exhibit an enhanced increasing CO2 absorption kinetics in the CO2 capture capacity from 37.2 wt % to 48.3 wt % without apparent degradation with increasing carbonation/calcination cycling up to 50 at 700 °C due to the strong self-reactivation effect of the mesoporous Ca-Al-O microstructure. Remarkable improvements in the CaO-CaCO3 conversion attained from the mesostructured Ca-Al-O composite can be explained using the concept combined with available mesoporous structure and Ca12Al14O33 phase content. However, a high Ca(2+)/Al(3+) =8:1 Ca-Al-O composite causes degradation because the pores become blocked and partial sintering induces CaO agglomeration.

CO2 sorbents with scaffold-like Ca-Al layered double hydroxides as precursors for CO2 capture at high temperatures.

A highly stable high-temperature CO2 sorbent consisting of scaffold-like Ca-rich oxides (Ca-Al-O) with rapid absorption kinetics and a high capacity is described. The Ca-rich oxides were prepared by annealing Ca-Al-NO3 layered double hydroxide (LDH) precursors through a sol-gel process with Al(O(i)P)3 and Ca(NO3)2 with Ca(2+)/Al(3+) ratios of 1:1, 2:1, 4:1, and 7:1. XRD indicated that only LDH powders were formed for Ca(2+)/Al(3+) ratios of 2:1. However, both LDH and Ca(OH)2 phases were produced at higher ratios. Both TEM and SEM observations indicated that the Ca-Al-NO3 LDHs displayed a scaffold-like porous structure morphology rather than platelet-like particles. Upon annealing at 600 °C, a highly stable porous network structure of the CaO-based Ca-Al-O mixed oxide (CAMO), composed of CaO and Ca12Al14O33, was still present. The CAMO exhibited high specific surface areas (up to 191 m(2)g(-1)) and a pore size distribution of 3-6 nm, which allowed rapid diffusion of CO2 into the interior of the material, inducing fast carbonation/calcination and enhancing the sintering-resistant nature over multiple carbonation/calcination cycles for CO2 absorption at 700 °C. Thermogravimetric analysis results indicated that a CO2 capture capacity of approximately 49 wt% could be obtained with rapid absorption from the porous 7:1 CAMO sorbents by carbonation at 700 °C for 5 min. Also, 94-98% of the initial CO2 capture capability was retained after 50 cycles of multiple carbonation/calcination tests. Therefore, the CAMO framework is a good isolator for preventing the aggregation of CaO particles, and it is suitable for long-term cyclic operation in high-temperature environments.