The Endocannabinoid System Alleviates Pain in a Murine Model of Cancer-Induced Bone Pain.

Metastatic breast cancer is prevalent worldwide, and one of the most common sites of metastasis is long bones. Of patients with disease, the major symptom is pain, yet current medications fail to adequately result in analgesic efficacy and present major undesirable adverse effects. In our study, we investigate the potential of a novel monoacylglycerol lipase (MAGL) inhibitor, MJN110, in a murine model of cancer-induced bone pain. Literature has previously demonstrated that MAGL inhibitors function to increase the endogenous concentrations of 2-arachydonylglycerol, which then activates CB1 and CB2 receptors to inhibit inflammation and pain. We demonstrate that administration of MJN110 significantly and dose dependently alleviates spontaneous pain behavior during acute administration compared with vehicle control. In addition, MJN110 maintains its efficacy in a chronic-dosing paradigm over the course of 7 days without signs of receptor sensitization. In vitro analysis of MJN110 demonstrated a dose-dependent and significant decrease in cell viability and proliferation of 66.1 breast adenocarcinoma cells to a greater extent than KML29, an alternate MAGL inhibitor, or the CB2 agonist JWH015. Chronic administration of the compound did not appear to affect tumor burden, as evidenced by radiograph or histologic analysis. Together, these data support the application for MJN110 as a novel therapeutic for cancer-induced bone pain. SIGNIFICANCE STATEMENT: Current standard of care for metastatic breast cancer pain is opioid-based therapies with adjunctive chemotherapy, which have highly addictive and other deleterious side effects. The need for effective, non-opioid-based therapies is essential, and harnessing the endogenous cannabinoid system is proving to be a new target to treat various types of pain conditions. We present a novel drug targeting the endogenous cannabinoid system that is effective at reducing pain in a mouse model of metastatic breast cancer to bone.

Methyl 2-(5,5-dimethyl-1,3,2-dioxa-borinan-2-yl)-4-nitro-benzoate.

The six-membered boronate ester ring of the title compound, C(13)H(16)BNO(6), adopts an envelope conformation with the C atom bearing the dimethyl substituents at the flap. The O-B-C-C torsion angles between the boronate group and the benzene ring are 72.5 (2) and 81.0 (2)°. The 4-nitro-benzoate unit adopts a slightly twisted conformation, with dihedral angles between the benzene ring and the nitrate and methyl ester groups of 17.5 (2) and 14.4 (3)°, respectively. In the crystal, inversion-related pairs of mol-ecules show weak π-π stacking inter-actions [centroid-centroid distance = 4.0585 (9) Šand inter-planar spacing = 3.6254 (7) Å].

6-Azido-3-O-benzyl-6-de-oxy-N,N-diethyl-1,2-O-isopropyl-idene-d-glycero-α-d-gluco-heptofuran-uronamide.

Reaction of 3-O-benzyl-1,2-O-isopropyl-idene-α-xylo-pentodialdo-1,4-furan-ose with N,N-diethyl-2-(dimethyl-sulfuranil-idene)acetamide gave stereoselectively an ep-oxy-amide, which was regioselectively opened by NaN(3) in dimethyl formamide to give the title compound, C(21)H(30)N(4)O(6). X-ray crystallography confirmed the relative stereochemistry of the title compound and the absolute configuration was determined by the use of d-glucose as the starting material. There are two mol-ecules in the asymmetric unit (Z' = 2). The crystal structure consists of two types of chains of O-H⋯O hydrogen-bonded mol-ecules running parallel to the b axis, with each mol-ecule acting as a donor and acceptor of one hydrogen bond.

Uricolytic enzymes in liver of the Dipnoan Protopterus aethiopicus.

The enzymes uricase, allantoinase, and allantoicase have been measured in liver preparations of the African lungfish Protopterus aethiopicus. The levels for these enzymes in lungfish liver suggest that the amount of urea formed in vivo in Protopterus via a uricolytic pathway may be greater than that derived via the Ornithine-urea cycle. The operation of a "purine cycle" in lungfish liver is proposed.

Caesarean delivery, immune function and inflammation in early life among Ecuadorian infants and young children.

Caesarean delivery has been linked to a number of inflammatory conditions in childhood and adolescence. Yet the mechanisms underlying these associations and their generalizability across contexts with different postnatal feeding and pathogenic exposures remain unclear. This study tests the association between delivery type and three measures of immune function, inflammation, morbidity and leukocyte proportions, in Ecuadorian infants and children aged 6 months to 2 years. Data were collected from mother-child pairs participating in a nationally representative health and nutrition survey Encuesta Nacional de Salud y Nutricion (ENSANUT-ECU) conducted in 2012. The analytic sample includes 828 mothers and infants with delivery information and measured biomarkers. Poisson regression models were used to examine the association between delivery type and markers of immune function, controlling for maternal and infant characteristics, including age, sex, sociodemographic characteristics and medical indications. 40.8% (n=338) of sample infants and children were delivered by caesarean. Compared to those born vaginally, infants born by caesarean were less likely to have elevated C-reactive protein (CRP) [CRP>2 mg/l; risk ratio (RR): 0.76, 95% confidence interval (CI): 0.58-1.00] and more likely to have illness symptoms (RR: 1.22, 95% CI: 1.01-1.46) and elevated basophils (RR: 1.83, 95% CI: 1.03-3.25). No other immune cell proportions differed by delivery type. The results suggest that differences in the perinatal exposures accompanying caesarean delivery may alter immune development and function, particularly in the inflammatory response to infection and in cells involved in the allergic response, across infancy and early childhood. Understanding the pathways linking perinatal exposures to immune development is important for preventing the development of inflammatory conditions.

Evidence suggesting persistence of nephritogenic immunopathologic mechanisms in patients receiving renal allografts.

Direct immunofluorescent (IF) examinations and elutions were performed on native kidneys and allografts of 24 patients undergoing renal transplantation. Immunoglobulins (Ig) were detected by IF on native kidneys of 12 of the 24; 11 of the 12 later had Ig localized to allograft glomeruli by direct IF. In addition, three other patients also developed Ig deposition on allograft glomeruli, although direct IF of native kidneys was negative. Elution studies indicated: (a) that linear Ig deposition on allograft glomeruli was the result of antiglomerular basement membrane (GBM) antibodies, (b) Ig localizing to allograft glomeruli in many of these patients was the result of persistent immunopathogenetic mechanisms existing at the time of allograft placement, and (c) occasionally, kidneys negative for Ig localization by direct IF contain elutable nephritogenic antibodies.

Concordance of haemoglobin A1c, blood pressure and C-reactive protein between children and their parents in Chinese households.

BACKGROUND: China has the world's highest diabetes prevalence, which along with hypertension and inflammation continues to grow particularly among children. Little is known about the strength of the association of these cardiometabolic risk factors between parents and their children; thus, the potential of household-based strategies to reduce risk is unknown. OBJECTIVES: The objective of the study is to examine the parent-child association for haemoglobin A1c (HbA1c), blood pressure (BP) and C-reactive protein (CRP) in a large, geographically diverse Chinese sample. METHODS: In 940 parent-child pairs (children aged 7-17 years) who participated in the 2009 China Health and Nutrition Survey, we measured each individual's HbA1c and CRP using fasting blood and BP. We used sex-specific random-effects linear regression to examine the parent-child association for these risk factors, accounting for within-family clustering. RESULTS: Child's HbA1c was positively associated with parental HbA1c. Beta coefficients ranged from 0.06 (95% CI 0.03-0.12) for father-daughter to 0.43 (95% CI 0.28-0.58) for mother-son pairs. We also detected a positive mother-daughter association for BP and positive father-child associations for CRP. CONCLUSION: The statistically significant parent-child association for HbA1c, BP and CRP in Chinese families suggests that household-based interventions could be useful for confronting the high rates of diabetes, hypertension and inflammation in China.

Acyl-CoA inhibition of hexokinase in rat and human skeletal muscle is a potential mechanism of lipid-induced insulin resistance.

There are strong correlations between impaired insulin-stimulated glucose metabolism and increased intramuscular lipid pools; however, the mechanism by which lipids interact with glucose metabolism is not completely understood. Long-chain acyl CoAs have been reported to allosterically inhibit liver glucokinase (hexokinase IV). The aim of the present study was to determine whether long-chain acyl CoAs inhibit hexokinase in rat and human skeletal muscle. At subsaturating glucose concentrations, 10 micromol/l of the three major long-chain acyl-CoA species in skeletal muscle, palmitoyl CoA (16:0), oleoyl CoA (18:1, n = 9), and linoleoyl CoA (18:2, n = 6), reduced hexokinase activity of rat skeletal muscle to 61 +/- 3, 66 +/- 7, and 57 +/- 5% of control activity (P < 0.005), respectively. The inhibition was concentration-dependent (P < 0.005) with 5 pmol/l producing near maximal inhibition. Human skeletal muscle hexokinase was also inhibited by long-chain acyl CoAs (5 pmol/l palmitoyl CoA decreased activity to 75 +/- 6% of control activity, P < 0.005). Inhibition of hexokinase in rat and human muscle by long-chain acyl CoAs was additive to the inhibition of hexokinase by glucose-6-phosphate (an allosteric inhibitor of hexokinase). This inhibition of skeletal muscle hexokinase by long-chain acyl CoA suggests that increases in intramuscular lipid metabolites could interact directly with insulin-mediated glucose metabolism in vivo by decreasing the rate of glucose phosphorylation and decreasing glucose-6-phosphate concentrations.

Quantitative hopanoid analysis enables robust pattern detection and comparison between laboratories.

Hopanoids are steroid-like lipids from the isoprenoid family that are produced primarily by bacteria. Hopanes, molecular fossils of hopanoids, offer the potential to provide insight into environmental transitions on the early Earth, if their sources and biological functions can be constrained. Semiquantitative methods for mass spectrometric analysis of hopanoids from cultures and environmental samples have been developed in the last two decades. However, the structural diversity of hopanoids, and possible variability in their ionization efficiencies on different instruments, have thus far precluded robust quantification and hindered comparison of results between laboratories. These ionization inconsistencies give rise to the need to calibrate individual instruments with purified hopanoids to reliably quantify hopanoids. Here, we present new approaches to obtain both purified and synthetic quantification standards. We optimized 2-methylhopanoid production in Rhodopseudomonas palustris TIE-1 and purified 2Me-diplopterol, 2Me-bacteriohopanetetrol (2Me-BHT), and their unmethylated species (diplopterol and BHT). We found that 2-methylation decreases the signal intensity of diplopterol between 2 and 34% depending on the instrument used to detect it, but decreases the BHT signal less than 5%. In addition, 2Me-diplopterol produces 10× higher ion counts than equivalent quantities of 2Me-BHT. Similar deviations were also observed using a flame ionization detector for signal quantification in GC. In LC-MS, however, 2Me-BHT produces 11× higher ion counts than 2Me-diplopterol but only 1.2× higher ion counts than the sterol standard pregnane acetate. To further improve quantification, we synthesized tetradeuterated (D4) diplopterol, a precursor for a variety of hopanoids. LC-MS analysis on a mixture of (D4)-diplopterol and phospholipids showed that under the influence of co-eluted phospholipids, the D4-diplopterol internal standard quantifies diplopterol more accurately than external diplopterol standards. These new quantitative approaches permit meaningful comparisons between studies, allowing more accurate hopanoid pattern detection in both laboratory and environmental samples.

Implications of iron deficiency/anemia on the classification of diabetes using HbA1c.

BACKGROUND/OBJECTIVES: Nonglycemic factors like iron deficiency (ID) or anemia may interfere with classification of diabetes and prediabetes using hemoglobin A1c (HbA1c). However, few population-based studies of diabetes in areas with endemic ID/anemia have been conducted. We aimed to determine how mutually exclusive categories of ID alone, anemia alone and iron-deficiency anemia (IDA) were each associated with prediabetes and diabetes prevalence using fasting blood glucose (FBG) versus HbA1c in a population-based study of adults with endemic ID/anemia. SUBJECTS/METHODS: We used data from the China Health and Nutrition Survey, a longitudinal, population-based study across 228 communities within nine provinces of China. This analysis included 7308 adults seen in the 2009 survey aged 18-75 years. We used descriptive and covariate-adjusted models to examine relative risk of prediabetes and diabetes using FBG alone, HbA1c alone, HbA1c and FBG, or neither (normoglycemia) by anemia alone, ID alone, IDA or normal iron/hemoglobin. RESULTS: Approximately 65% of individuals with diabetes in our sample were concordantly classified with diabetes using both FBG and HbA1c, while 35% had a discordant diabetes classification: they were classified using either FBG or HbA1c, but not both. Fewer participants with ID alone versus normal iron/hemoglobin were classified with diabetes using HbA1c only. From covariate-adjusted, multinomial regression analyses, the adjusted prevalence of prediabetes using HbA1c only was 22% for men with anemia alone, but 13% for men with normal iron/hemoglobin. In contrast, the predicted prevalence of prediabetes using HbA1c only was 8% for women with ID alone, compared with 13% for women with normal iron/hemoglobin. CONCLUSIONS: These findings suggest potential misclassification of diabetes using HbA1c in areas of endemic ID/anemia. Estimating diabetes prevalence using HbA1c may result in under-diagnosis in women with ID and over-diagnosis in men with anemia.

Muscle long-chain acyl CoA esters and insulin resistance.

A common observation in animal models and in humans is that accumulation of muscle triglyceride is associated with the development of insulin resistance. In animals, this is true of genetic models of obesity and nutritional models of insulin resistance generated by high-fat feeding, infusion of lipid, or infusion of glucose. Although there is a strong link between the accumulation of triglycerides (TG) in muscle and insulin resistance, it is unlikely that TG are directly involved in the generation of muscle insulin resistance. There are now other plausible mechanistic links between muscle lipid metabolites and insulin resistance, in addition to the classic substrate competition proposed by Randle's glucose-fatty acid cycle. The first step in fatty acid metabolism (oxidation or storage) is activation to the long-chain fatty acyl CoA (LCACoA). This review covers the evidence suggesting that cytosolic accumulation of this active form of lipid in muscle can lead to impaired insulin signaling, impaired enzyme activity, and insulin resistance, either directly or by conversion to other lipid intermediates that alter the activity of key kinases and phosphatases. Actions of fatty acids to bind specific nuclear transcription factors provide another mechanism whereby different lipids could influence metabolism.

Mechanical ventilation increases microvascular permeability in oleic acid-injured lungs.

The pulmonary pathology for which a patient receives ventilatory support may increase the risk of developing barotrauma, because an underlying disease process may weaken the vasculature and render the lung more susceptible to damage by mechanical ventilation. We determined the response of isolated young rabbit lungs to mechanical ventilation after oleic acid (OA) injury. New Zealand White rabbits (0.7-1.3 kg) were anesthetized with pentobarbital sodium (30 mg/kg), tracheotomized, and exsanguinated. The heart and lungs were isolated and perfused with autologous blood at a constant flow. The capillary filtration coefficient (Kf,c, in ml.min-1.cmH2O-1.100 g wet wt-1) and pulmonary arterial (Ppa) and venous pressures were determined before and 30 and 60 min after oleic acid administration (OA group; 0.2 ml into the venous reservoir), ventilation alone (Vent group; peak inspiratory pressure = 25 cmH2O), or oleic acid combined with ventilation (OA + Vent group). Ppa transiently increased by 4.21 +/- 0.822 cmH2O after OA administration but then returned to approximately control values. Baseline Kf,c values for OA (0.288 +/- 0.042), Vent (0.296 +/- 0.035), or OA + Vent (0.276 +/- 0.028) groups were not significantly different from each other. Kf,c after either OA administration (0.45 +/- 0.066) or Vent (0.35 +/- 0.75) were not significantly different from each other or from baseline measurements. In the group ventilated after OA administration (OA + Vent), Kf,c (0.883 +/- 0.148) increased significantly from baseline (P less than 0.001) and was significantly different from all other treatment groups. We conclude that the combination of minimal OA injury and ventilation was more deleterious to the lung than either one alone.

Studies of visual synthesis: integration of fragments into forms.

On each of a series of trials, subjects indicated whether two sequential visual forms were the same or different. The first form was presented on some trials as a whole within a square frame and on others in fragments--with its perimeter distributed over two or three frames. The subject was instructed to visualize the first form as a whole in any case, and study time (ST) for the form was recorded. The same/different reaction time (RT) for the second, test, form (always presented in one frame) was also recorded. Experiment 1 revealed that variables affecting ST had little effect on RT and indicated that subjects can process fragments of forms in parallel. Experiment 2 demonstrated that the representation encoded from fragments differed from one generated from long-term memory. Whereas the first experiments used wholes as test stimuli, in Experiments 3 and 4 on some trials, fragments were used. In Experiment 3, RT was facilitated when a test fragment coincided with an intact fragment of the first stimulus. This indicated that first stimuli were not encoded as complete wholes. In Experiment 4, irregular forms were used as stimuli, and the RT data departed from predictions of the parallel model. Taken as a whole, the results place constraints on the codes produced by constructive processes acting to synthesize fragments into wholes.

Triglycerides, fatty acids and insulin resistance--hyperinsulinemia.

There is now much interest in the mechanisms by which altered lipid metabolism might contribute to insulin resistance as is found in Syndrome X or in Type II diabetes. This review considers recent evidence obtained in animal models and its relevance to humans, and also likely mechanisms and strategies for the onset and amelioration of insulin resistance. A key tissue for development of insulin resistance is skeletal muscle. Animal models of Syndrome X (eg high fat fed rat) exhibit excess accumulation of muscle triglyceride coincident with development of insulin resistance. This seems to also occur in humans and several studies demonstrate increased muscle triglyceride content in insulin resistant states. Recently magnetic resonance spectroscopy has been used to demonstrate that at least some of the lipid accumulation is inside the muscle cell (myocyte). Factors leading to this accumulation are not clear, but it could derive from elevated circulating free fatty acids, basal or postprandial triglycerides, or reduced muscle fatty acid oxidation. Supporting a link with adipose tissue metabolism, there appears to be a close association of muscle and whole body insulin resistance with the degree of abdominal obesity. While causal relationships are still to be clearly established, there are now quite plausible mechanistic links between muscle lipid accumulation and insulin resistance, which go beyond the classic Randle glucose-fatty acid cycle. In animal models, dietary changes or prior exercise which reduce muscle lipid accumulation also improve insulin sensitivity. It is likely that cytosolic accumulation of the active form of lipid in muscle, the long chain fatty acyl CoAs, is involved, leading to altered insulin signalling or enzyme activities (eg glycogen synthase) either directly or via chronic activation of mediators such as protein kinase C. Unless there is significant weight loss, short or medium term dietary manipulation does not alter insulin sensitivity as much in humans as in rodent models, and there is considerable interest in pharmacological intervention. Studies using PPARgamma receptor agonists, the thiazolidinediones, have supported the principle that reduced muscle lipid accumulation is associated with increased insulin sensitivity. Other potent systemic lipid-lowering agents such as PPARalpha receptor agonists (eg fibrates) or antilipolytic agents (eg nicotinic acid analogues) might improve insulin sensitivity but further work is needed, particularly to clarify implications for muscle metabolism. In conclusion, evidence is growing that excess muscle and liver lipid accumulation causes or exacerbates insulin resistance in Syndrome X and in Type II diabetes; development of strategies to prevent this seem very worthwhile.

The role of lipids in the pathogenesis of muscle insulin resistance and beta cell failure in type II diabetes and obesity.

This review considers evidence for, and putative mechanisms of, lipid-induced muscle insulin resistance. Acute free fatty acid elevation causes muscle insulin resistance in a few hours, with similar muscle lipid accumulation as accompanies more prolonged high fat diet-induced insulin resistance in rodents. Although causal relations are not as clearcut in chronic human insulin resistant states such as obesity and type 2 diabetes, it is now recognised that muscle lipids also accumulate in these states. The classic Randle glucose-fatty acid cycle is only one of a number of mechanisms by which fatty acids might influence muscle glucose metabolism and insulin action. A key factor is seen to be accumulation of muscle long chain acyl CoAs, which could alter insulin action via several mechanisms including chronic activation of protein kinase C isoforms or ceramide accumulation. These interactions are fundamental to understanding metabolic effects of new insulin "sensitizers", e.g. thiazolidinediones, which alter lipid metabolism and improve muscle insulin sensitivity in insulin resistant states. Recent work has also pointed to a possible role of lipids in beta cell deterioration ("lipotoxicity") associated with type 2 diabetes.

Salt and blood pressure: the triangular hypothesis.

If an unselected sample includes individuals whose blood pressure is sensitive to their salt intake and individuals whose blood pressure is not sensitive, then the superposition of these two sub-populations in a scatterplot of individuals' blood pressures against their salt intakes could give a triangular distribution. The non-correlation in the insensitives would obscure the correlation expected in the sensitives. This hypothesis justifies truncation of such data to test for correlation between blood pressure and salt intake among only the individuals in the higher range of blood pressures observed. No criterion of salt sensitivity is needed. The analysis should succeed if salt intake makes a major contribution to hypertension and would be improved if other putative causes were factored out.

Practical applications of averages and differences of Friedel opposites.

The practical use of the average and difference intensities of Friedel opposites at different stages of structure analysis has been investigated. It is shown how these values may be properly and practically used at the stage of space-group determination. At the stage of least-squares refinement, it is shown that increasing the weight of the difference intensities does not improve their fit to the model. The correct form of the coefficients for a difference electron-density calculation is given. In the process of structure validation, it is further shown that plots of the observed and model difference intensities provide an objective method to evaluate the fit of the data to the model and to reveal insufficiencies in the intensity measurements. As a further tool for the validation of structure determinations, the use of the Patterson functions of the average and difference intensities has been investigated and their clear advantage demonstrated.

In pursuit of individualised margins for prostate cancer patients undergoing image-guided radiotherapy: the effect of body mass index on intrafraction prostate motion.

AIMS: To analyse the relationship between body mass index (BMI) and intrafraction displacement in patients undergoing prostate cancer image-guided radiotherapy. MATERIALS AND METHODS: An analysis was carried out on 151 prostate cancer patients treated with radical dose radiotherapy between January 2007 and March 2009. Patients had their height, weight and daily intrafraction prostate displacement data collected prospectively during fiducial marker image-guided radiotherapy with orthogonal imaging. For each of anterior-posterior, left-right and superior-inferior axes, a univariable linear regression analysis was carried out with the individual patient standard deviation of shift as the response variable and BMI as a continuous explanatory variable. RESULTS: Displacement measurements were recorded from 4764 pre- and post-treatment image sets. Patients were grouped according to BMI as normal weight (24%), overweight (52%), obese (18%), severely obese (3%) or morbidly obese (3%). For intrafraction displacement, a one unit increase in BMI affected the standard deviation of shift by: anterior-posterior -0.02 (95% confidence interval -0.040 to 0.000), left-right -0.006 (95% confidence interval -0.020 to 0.008) and superior-inferior -0.020 (95% confidence interval -0.037 to -0.003). CONCLUSIONS: Our data indicate that patients with a higher BMI have less intrafraction displacement of the prostate in the superior-inferior dimension compared with patients with a lower BMI. This has implications for individualised treatment margins for future prostate cancer patients undergoing image-guided radiotherapy. Further study is recommended.