Inhibition of DAGLβ as a therapeutic target for pain in sickle cell disease

Sickle cell disease (SCD) is the most common inherited disease. Pain is a key morbidity of SCD and opioids are the main treatment but their side effects emphasize the need for new analgesic approaches. Humanized transgenic mouse models have been instructive in understanding the pathobiology of SCD and mechanisms of pain. Homozygous (HbSS) Berkley mice express >99% human sickle hemoglobin and several features of clinical SCD including hyperalgesia. Previously, we reported that the endocannabinoid 2-arachidonoylglycerol (2-AG) is a precursor of the pro-nociceptive mediator prostaglandin E2-glyceryl ester (PGE2-G) which contributes to hyperalgesia in SCD. We now demonstrate the causal role of 2-AG in hyperalgesia in sickle mice. Hyperalgesia in HbSS mice correlated with elevated levels of 2-AG in plasma, its synthesizing enzyme diacylglycerol lipase β (DAGLβ) in blood cells, and with elevated levels of PGE2 and PGE2-G, pronociceptive derivatives of 2-AG. A single intravenous injection of 2-AG produced hyperalgesia in non-hyperalgesic HbSS mice, but not in control (HbAA) mice expressing normal human HbA. JZL184, an inhibitor of 2-AG hydrolysis, also produced hyperalgesia in non-hyperalgesic HbSS or hemizygous (HbAS) mice, but did not influence hyperalgesia in hyperalgesic HbSS mice. Systemic and intraplantar administration of KT109, an inhibitor of DAGLβ, decreased mechanical and heat hyperalgesia in HbSS mice. The decrease in hyperalgesia was accompanied by reductions in 2-AG, PGE2 and PGE2-G in the blood. These results indicate that maintaining the physiological level of 2-AG in the blood by targeting DAGLβ may be a novel and effective approach to treat pain in SCD

Determining the Phosphorus Release Curve for Sunphase HT Phytase from 250 to 2,000 FTU/kg in Nursery Pig Diets

A total of 280 pigs (DNA 241 × 600; initially 22.9 ± 0.52 lb BW) were used in a 21-d growth study to determine the available P (aP) release curve for Sunphase HT phytase (Wuhan Sunhy Biology Co., Ltd.; Wuhan, P.R. China). At approximately 19 d of age, pigs were weaned, randomly allotted to pens, and fed common starter diets. Pigs were blocked by average pen body weight (BW) and randomly allotted to 1 of 7 dietary treatments on d 21 post-weaning, considered d 0 of the study. Dietary treatments were derived from a single basal diet, and ingredients including phytase, monocalcium P, limestone, and sand were added to create the treatment diets. Treatments included 3 diets containing increasing (0.11, 0.19, and 0.27% aP) inorganic P from monocalcium P, or 4 diets with increasing phytase (250, 500, 1,000, or 2,000 FTU/kg) added to the diet containing 0.11% aP. All diets were corn-soybean meal-canola meal-based and were formulated to contain 1.24% SID Lys and an analyzed Ca:P ratio of 1.10:1. Prior to the beginning of the study, all pigs were fed a diet containing 0.11% aP for a 3-d period (d 18 to 21 post-weaning). At the conclusion of the study, 1 pig, closest to the mean weight of each pen, was euthanized and the right fibula, rib, and metacarpal were collected to determine bone ash and density. For the overall experimental period, pigs fed increasing levels of aP from inorganic P had improved (linear, P ≤ 0.014) ADG, F/G, and final BW. Similarly, pigs fed increasing phytase had increased (linear, P ≤ 0.011) ADG and final BW as well as improved (quadratic, P = 0.017) F/G. For fibula bone ash weight and percentage bone ash, rib bone ash weight and bone density, and all metacarpal bone properties, pigs fed increasing levels of aP from inorganic P exhibited a linear improvement (P ≤ 0.019), with a quadratic response (P ≤ 0.030) for fibula bone density and rib percentage bone ash. Additionally, pigs fed increasing phytase had increased (P \u3c 0.05) bone ash weight, percentage bone ash, and bone density in either a linear or quadratic fashion depending on the bone analyzed. The available P release curve generated for Sunphase HT for percentage bone ash combining values from right fibula, rib, and metacarpal is: aP = (0.360 × FTU) ÷ (2,330.250 + FTU)

Amyloid Precursor Protein and Proinflammatory Changes Are Regulated in Brain and Adipose Tissue in a Murine Model of High Fat Diet-Induced Obesity

Background: Middle age obesity is recognized as a risk factor for Alzheimer’s disease (AD) although a mechanistic linkage remains unclear. Based upon the fact that obese adipose tissue and AD brains are both areas of proinflammatory change, a possible common event is chronic inflammation. Since an autosomal dominant form of AD is associated with mutations in the gene coding for the ubiquitously expressed transmembrane protein, amyloid precursor protein (APP) and recent evidence demonstrates increased APP levels in adipose tissue during obesity it is feasible that APP serves some function in both disease conditions. Methodology/Principal Findings: To determine whether diet-induced obesity produced proinflammatory changes and altered APP expression in brain versus adipose tissue, 6 week old C57BL6/J mice were maintained on a control or high fat diet for 22 weeks. Protein levels and cell-specific APP expression along with markers of inflammation and immune cell activation were compared between hippocampus, abdominal subcutaneous fat and visceral pericardial fat. APP stimulation-dependent changes in macrophage and adipocyte culture phenotype were examined for comparison to the in vivo changes. Conclusions/Significance: Adipose tissue and brain from high fat diet fed animals demonstrated increased TNF-a and microglial and macrophage activation. Both brains and adipose tissue also had elevated APP levels localizing to neurons and macrophage/adipocytes, respectively. APP agonist antibody stimulation of macrophage cultures increased specific cytokin

Fatty Acid Biosynthesis Inhibition Increases Reduction Potential in Neuronal Cells under Hypoxia

Recently, we have reported a novel neuronal specific pathway for adaptation to hypoxia through increased fatty acid (FA) biosynthesis (FAS) followed by esterification into lipids. However, the biological role of this pathway under hypoxia remains to be elucidated. In the presented study, we have tested our hypothesis that activation of FAS maintains reduction potential and reduces lactoacidosis in neuronal cells under hypoxia. To address this hypothesis, we measured the effect of FAS inhibition on NADH2+/NAD+ and NADPH2+/NADP+ ratios, and lactic acid levels in neuronal SH-SY5Y cells exposed to normoxic and hypoxic conditions. FAS inhibitors, TOFA (inhibits Acetyl-CoA carboxylase) and cerulenin (inhibits FA synthase), increased NADH2+/NAD+ and NADPH2+/NADP+ ratios under hypoxia. Further, FAS inhibition increased lactic acid under both normoxic and hypoxic conditions, and caused cytotoxicity under hypoxia but not normoxia. These results indicate that FA may serve as hydrogen acceptors under hypoxia, thus supporting oxidation reactions including anaerobic glycolysis. These findings may help to identify a radically different approach to attenuate hypoxia related pathophysiology in the nervous system including stroke

PFOA and PFOS Are Generated from Zwitterionic and Cationic Precursor Compounds During Water Disinfection with Chlorine or Ozone

Perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) are anionic organic pollutants, which are widespread in the environment. They have become a global concern due to their persistence in the environment as well as their toxicity and bioaccumulative properties. In this study, we demonstrate that PFOA, PFOS, or both are produced from a group of four zwitterionic/cationic polyfluoroalkyl amide (FA) and sulfonamide (FS) compounds during conventional drinking-water disinfection with chlorine or ozone. FA compounds were readily degraded by chlorine and converted primarily to PFOA, likely by a Hofmann-type rearrangement. FS compounds were much less reactive toward chlorine; the generation of PFOS from the FSs was not significant. All four FA and FS compounds were degraded rapidly during ozonation, generating PFOA, PFOS, and a number of infrequently reported products for which chemical structures were either confirmed or tentatively proposed using high-resolution mass spectrometry. FSs generated both PFOS and PFOA during ozonation with the yield of PFOA even higher than that from the FAs. The results of this study may provide important insight into the degradation mechanisms of FAs and FSs and shed light on their contribution to the secondary formation of PFOA and PFOS in natural and engineered systems

APP, CD45, and CD68 immunoreactivity increased in the visceral (pericardial) fat from high fat versus control diet fed mice with no robust change in Aβ immunoreactivity.

<p>C57BL6/J mice at 6 weeks of age and weight matched were fed, <i>ad libitum</i>, a control (5.5% fat/weight) or high fat (21.2% fat/weight) diet for 22 weeks. Visceral pericardial adipose tissue was collected, immersion fixed, sectioned, and immunostained using anti-APP, Aβ, CD45 and CD68 antibodies and antibody binding visualized using Vector VIP as the chromogen. Representative images from 12 animals per condition are shown.</p

Mitochondrial Lipid Abnormality and Electron Transport Chain Impairment in Mice Lacking α-Synuclein

The presynaptic protein α-synuclein, implicated in Parkinson disease (PD), binds phospholipids and has a role in brain fatty acid (FA) metabolism. In mice lacking α-synuclein (Snca(−/−)), total brain steady-state mass of the mitochondria-specific phospholipid, cardiolipin, is reduced 22% and its acyl side chains show a 51% increase in saturated FAs and a 25% reduction in essential n-6, but not n-3, polyunsaturated FAs. Additionally, 23% reduction in phosphatidylglycerol content, the immediate biosynthetic precursor of cardiolipin, was observed without alterations in the content of other brain phospholipids. Consistent with these changes, more ordered lipid head group and acyl chain packing with enhanced rotational motion of diphenylhexatriene (DPH) about its long axis were demonstrated in time-resolved DPH fluorescence lifetime experiments. These abnormalities in mitochondrial membrane properties were associated with a 15% reduction in linked complex I/III activity of the electron transport chain, without reductions in mitochondrial number, complex II/III activity, or individual complex I, II, III, or IV activity. Reduced complex I activity is thought to be a critical factor in the development of PD. Thus, altered membrane composition and structure and impaired complex I/III function in Snca(−/−) brain suggest a relationship between α-synuclein's role in brain lipid metabolism, mitochondrial function, and PD