Sublethal Hyperthermia Transiently Disrupts Cortisol Steroidogenesis in Adrenocortical Cells.

Primary aldosteronism is the most common cause of secondary hypertension. The first-line treatment adrenalectomy resects adrenal nodules and adjacent normal tissue, limiting suitability to those who present with unilateral disease. Use of thermal ablation represents an emerging approach as a possible minimally invasive therapy for unilateral and bilateral disease, to target and disrupt hypersecreting aldosterone-producing adenomas, while preserving adjacent normal adrenal cortex. To determine the extent of damage to adrenal cells upon exposure to hyperthermia, the steroidogenic adrenocortical cell lines H295R and HAC15 were treated with hyperthermia at temperatures between 37 and 50°C with the effects of hyperthermia on steroidogenesis evaluated following stimulation with forskolin and ANGII. Cell death, protein/mRNA expression of steroidogenic enzymes and damage markers (HSP70/90), and steroid secretion were analyzed immediately and 7 days after treatment. Following treatment with hyperthermia, 42°C and 45°C did not induce cell death and were deemed sublethal doses while ≥50°C caused excess cell death in adrenal cells. Sublethal hyperthermia (45°C) caused a significant reduction in cortisol secretion immediately following treatment while differentially affecting the expression of various steroidogenic enzymes, although recovery of steroidogenesis was evident 7 days after treatment. As such, sublethal hyperthermia, which occurs in the transitional zone during thermal ablation induces a short-lived, unsustained inhibition of cortisol steroidogenesis in adrenocortical cells in vitro.

Major disparities in patient-reported adherence compared to objective assessment of adherence using mass spectrometry: A prospective study in a tertiary-referral hypertension clinic.

AIM: Many challenges exist in determining true rates of adherence to antihypertensive medications among individuals in a clinic setting. For the first time, we aimed to compare patient-reported antihypertensive adherence with objective evidence using mass spectrometry spot urinalysis in a tertiary referral clinic setting. METHODS: A prospective observational single-centre cohort study was performed in a tertiary referral hypertension clinic, encompassing antihypertensive initiation and persistence. Patients were referred with apparent treatment-resistant hypertension or for suspected secondary causes. Participants completed a self-reported assessment of antihypertensive adherence and provided a spot urine sample. The presence of antihypertensive medications and/or their respective metabolites was evaluated using high-performance liquid chromatography tandem mass spectrometry. Patients were determined to be adherent if they demonstrated both self-reported adherence and objective mass spectrometry evidence. RESULTS: Of all 105 eligible participants initially recruited, 73 (69.5%) met the eligibility criteria. Only 27.4% (95% confidence interval 0.2-0.4) of participants demonstrated true adherence to their self-reported antihypertensives, despite 75.3% (0.6-0.8) reporting adherence. Greatest medication adherence was achieved with angiotensin II receptor blockers (61%), with calcium-channel blockers and mineralocorticoid antagonists demonstrating least adherence (38%). CONCLUSION: In patients attending a tertiary hypertension clinic, the combined use of spot urine mass spectrometry and self-reporting identifies higher rates of nonadherence when compared to either modality alone. Both techniques should be combined for more accurate detection of medication adherence.

Antinociceptive Effects of the GPR55 Antagonist CID16020046 Injected into the Rat Anterior Cingulate Cortex.

The G-protein coupled receptor, GPR55, modulates nociceptive processing. Given the expression of GPR55 in the anterior cingulate cortex (ACC), a key brain region involved in the cognitive and affective dimensions of pain, the present study tested the hypothesis that GPR55 signalling in the ACC facilitates inflammatory pain behaviour in rats. The expression of GPR55 in the ACC was confirmed by both western blotting and immunostaining, with evidence for neuronal localisation. Microinjection of the selective GPR55 antagonist CID16020046 into the ACC of adult male Sprague-Dawley rats significantly reduced second phase formalin-evoked nociceptive behaviour compared with vehicle-treated controls. CID16020046 administration was associated with a reduction in phosphorylation of extracellular signal-regulated kinase (ERK), a downstream target of GPR55 activation, in the ACC. Intra-ACC administration of CID16020046 prevented the formalin-induced increases in expression of mRNA coding for the immediate early gene and marker of neuronal activity, c-Fos, in the ipsilateral dorsal horn of the spinal cord. Intra-plantar injection of formalin reduced tissue levels of the endogenous GPR55 ligand 2-arachidonoyl-sn-glycero-3-phosphoinositol (2-AGPI) in the ACC, likely reflecting its increased release/utilisation. These data suggest that endogenous activation of GPR55 signalling and increased ERK phosphorylation in the ACC facilitates inflammatory pain via top-down modulation of descending pain control.

Measuring adherence to therapy in apparent treatment-resistant hypertension: a feasibility study in Irish primary care.

BACKGROUND: Apparent treatment-resistant hypertension (aTRH) is defined as uncontrolled blood pressure (BP) in patients taking three or more antihypertensive medications. Some patients will have true treatment-resistant hypertension, some undiagnosed secondary hypertension, while others have pseudo-resistance. Pseudo-resistance occurs when non-adherence to medication, white-coat hypertension (WCH), lifestyle, and inadequate drug dosing are responsible for the poorly controlled BP. AIM: To examine the feasibility of establishing non-adherence to medication, for the first time in primary care, using mass spectrometry urine analysis. Operationalisation would be established by at least 50% of patients participating and 95% of samples being suitable for analysis. Clinical importance would be confirmed by >10% of patients being non-adherent. DESIGN AND SETTING: Eligible patients with aTRH (n = 453) in 15 university research-affiliated Irish general practices were invited to participate. METHOD: Participants underwent mass spectrometry urine analysis to test adherence and ambulatory BP monitoring (ABPM) to examine WCH. RESULTS: Of the eligible patients invited, 52% (n = 235) participated. All 235 urine samples (100%) were suitable for analysis: 174 (74%) patients were fully adherent, 56 (24%) partially adherent, and five (2%) fully non-adherent to therapy. A total of 206 patients also had ABPM, and in total 92 (45%) were categorised as pseudo-resistant. No significant associations were found between adherence status and patient characteristics or drug class. CONCLUSION: In patients with aTRH, the authors have established that it is feasible to examine non-adherence to medications using mass spectrometry urine analysis. One in four patients were found to be partially or fully non-adherent. Further research on how to incorporate this approach into individual patient consultations and its associated cost-effectiveness is now appropriate.

Medication adherence for resistant hypertension: Assessing theoretical predictors of adherence using direct and indirect adherence measures.

OBJECTIVES: This study examined theoretical predictors of long-term medication adherence (i.e., treatment-related beliefs, coherence of beliefs from experience with medication, habit strength, and pill burden) for patients with apparent treatment-resistant hypertension in primary care, using a composite adherence score derived from direct and indirect measures (i.e., prescription refill, self-report, and bioanalytical assays of urine). DESIGN: Cross-sectional study. METHODS: Individual patient records were screened for prescription refill adherence. Patients provided a urine sample for adherence screening and completed a battery of psychometric scales, including two self-report adherence measures (N = 204). Convergence of adherence measures was assessed, a composite adherence score was calculated, and hierarchical multiple regression was used to examine the role of theoretical predictors of adherence. RESULTS: Non-adherence estimates ranged from 20.3 to 41.1%, depending on the assessment method used. Associations among adherence measures were weak to moderate (ρ = .00-.53). Medication-taking habit strength was the strongest predictor of adherence, explaining 19% incremental variance in adherence beyond treatment-related beliefs. Beliefs and coherence did not predict adherence, even for patients with weaker habits. Pill burden was not associated with habit strength or adherence for this sample. CONCLUSIONS: Associations among unique adherence measures were weak overall, providing further evidence that multiple measures are necessary to accurately assess adherence. Habit strength is a key predictor of adherence for chronic conditions. Both habit strength and pill burden represent important intervention targets for improving long-term medication adherence. Longitudinal inception studies are needed to properly test Common-Sense Model propositions and elucidate the role of beliefs, coherence, and habits in predicting adherence at various stages of the chronic illness trajectory. Statement of contribution What is already known on this subject? Non-adherence to antihypertensives is a leading cause of apparent treatment-resistant hypertension (aTRH). Behaviour maintenance (vs. initiation) factors may be more predictive of long-term adherence. What does this study add? Associations among direct and indirect measures of adherence are generally weak. Habit strength is the strongest predictor of long-term adherence for aTRH in primary care. Inception studies are needed to further validate Common-Sense Model propositions.

Plasma N-acylethanolamine and endocannabinoid levels in burning mouth syndrome: Potential role in disease pathogenesis.

OBJECTIVE: The objective was to measure endocannabinoid (eCB) ligands and non-cannabinoid N-acylethanolamine (NAE) molecules in plasma from individuals with burning mouth syndrome (BMS) and to determine whether plasma eCB/NAE levels correlated with pain, inflammation and depressive symptomatology in this cohort. STUDY DESIGN: Plasma content of the eCBs, anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG), and the NAE molecules, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) were assessed in healthy subjects (n = 8) and in a cohort of newly diagnosed BMS patients (n = 9) using liquid chromatography-tandem mass spectrometry. Plasma eCBs and NAE profiles were correlated with self-rated oral cavity pain intensities, depressive symptomatology and plasma IL-8 levels. RESULTS: Plasma levels of PEA, but not OEA, AEA or 2-AG, were significantly elevated in patients with BMS, when compared to plasma from healthy individuals. Plasma PEA, OEA and AEA levels correlated with depressive symptomatology. CONCLUSIONS: This is the first evidence to indicate that circulating eCB/NAE levels are altered in BMS.

Characterisation of peroxisome proliferator-activated receptor signalling in the midbrain periaqueductal grey of rats genetically prone to heightened stress, negative affect and hyperalgesia.

The stress-hyperresponsive Wistar-Kyoto (WKY) rat strain exhibits a hyperalgesic phenotype and is a useful genetic model for studying stress-pain interactions. Peroxisome proliferator-activated receptor (PPAR) signalling in the midbrain periaqueductal grey (PAG) modulates pain. This study characterised PPAR signalling in the PAG of WKY rats exposed to the formalin test of inflammatory pain, versus Sprague-Dawley (SD) controls. Formalin injection reduced levels of the endogenous PPAR ligands N-palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA) in the lateral(l) PAG of SD rats, but not WKY rats which exhibited higher levels of these analytes compared with formalin-injected SD counterparts. Levels of mRNA coding for fatty acid amide hydrolase (FAAH; catabolises PEA and OEA) were lower in the lPAG of WKY versus SD rats. PPARγ mRNA and protein levels in the lPAG were higher in saline-treated WKY rats, with PPARγ protein levels reduced by formalin treatment in WKY rats only. In the dorsolateral(dl) or ventrolateral(vl) PAG, there were no effects of formalin injection on PEA or OEA levels but there were some differences in levels of these analytes between saline-treated WKY and SD rats and some formalin-evoked alterations in levels of PPARα, PPARγ or FAAH mRNA in WKY and/or SD rats. Pharmacological blockade of PPARγ in the lPAG enhanced formalin-evoked nociceptive behaviour in WKY, but not SD, rats. These data indicate differences in the PPAR signalling system in the PAG of WKY versus SD rats and suggest that enhanced PEA/OEA-mediated tone at PPARγ in the lPAG may represent an adaptive mechanism to lower hyperalgesia in WKY rats.

N-palmitoylethanolamide in the anterior cingulate cortex attenuates inflammatory pain behaviour indirectly via a CB1 receptor-mediated mechanism.

The neural substrates and mechanisms mediating the antinociceptive effects of the endogenous bioactive lipid, N-palmitoylethanolamide (PEA), require further investigation. We investigated the effects of exogenous PEA administration into the anterior cingulate cortex (ACC), an important brain region linked with cognitive and affective modulation of pain, on formalin-evoked nociceptive behaviour in rats. Potential involvement of peroxisome proliferator-activated receptor isoforms (PPAR) α and γ or endocannabinoid-mediated entourage effects at cannabinoid1 (CB1) receptors or transient receptor potential subfamily V member 1 (TRPV1) in mediating the effects of PEA was also investigated. Intra-ACC administration of PEA significantly attenuated the first and early second phases of formalin-evoked nociceptive behaviour. This effect was attenuated by the CB1 receptor antagonist AM251, but not by the PPARα antagonist GW6471, the PPARγ antagonist GW9662, or the TRPV1 antagonist 5'-iodo resiniferatoxin. All antagonists, administered alone, significantly reduced formalin-evoked nociceptive behaviour, suggesting facilitatory/permissive roles for these receptors in the ACC in inflammatory pain. Post-mortem tissue analysis revealed a strong trend for increased levels of the endocannabinoid anandamide in the ACC of rats that received intra-ACC PEA. Expression of c-Fos, a marker of neuronal activity, was significantly reduced in the basolateral nucleus of the amygdala, but not in the central nucleus of the amygdala, the rostral ventromedial medulla or the dorsal horn of the spinal cord. In conclusion, these data indicate that PEA in the ACC can reduce inflammatory pain-related behaviour, possibly via AEA-induced activation of CB1 receptors and associated modulation of neuronal activity in the basolateral amygdala.

Ptaquiloside in Irish Bracken Ferns and Receiving Waters, with Implications for Land Managers.

Ptaquiloside, along with other natural phytotoxins, is receiving increased attention from scientists and land use managers. There is an urgent need to increase empirical evidence to understand the scale of phytotoxin mobilisation and potential to enter into the environment. In this study the risk of ptaquiloside to drinking water was assessed by quantifying ptaquiloside in the receiving waters at three drinking water abstraction sites across Ireland and in bracken fronds surrounding the abstraction sites. We also investigated the impact of different management regimes (spraying, cutting and rolling) on ptaquiloside concentrations at plot-scale in six locations in Northern Ireland, UK. Ptaquiloside concentrations were determined using recent advances in the use of LC-MS for the detection and quantification of ptaquiloside. The results indicate that ptaquiloside is present in bracken stands surrounding drinking water abstractions in Ireland, and ptaquiloside concentrations were also observed in the receiving waters. Furthermore, spraying was found to be the most effective bracken management regime observed in terms of reducing ptaquiloside load. Increased awareness is vital on the implications of managing land with extensive bracken stands.

Microinjection of 2-arachidonoyl glycerol into the rat ventral hippocampus differentially modulates contextually induced fear, depending on a persistent pain state.

The endogenous cannabinoid (endocannabinoid) system plays a key role in the modulation of aversive and nociceptive behaviour. The components of the endocannabinoid system are expressed throughout the hippocampus, a brain region implicated in both conditioned fear and pain. In light of evidence that pain can impact on the expression of fear-related behaviour, and vice versa, we hypothesised that exogenous administration of the endocannabinoid 2-arachidonoyl glycerol (2-AG) into the ventral hippocampus (vHip) would differentially regulate fear responding in the absence vs. the presence of formalin-evoked nociceptive tone. Fear-conditioned rats showed significantly increased freezing and a reduction in formalin-evoked nociceptive behaviour upon re-exposure to a context previously paired with footshock. Bilateral microinjection of 2-AG into the vHip significantly reduced contextually induced freezing in non-formalin-treated rats, and reduced formalin-evoked nociceptive behaviour in non-fear-conditioned rats. In contrast, 2-AG microinjection had no effect on fear responding in formalin-treated rats, and no effect on nociceptive behaviour in fear-conditioned rats. The inhibitory effect of 2-AG on fear-related behaviour, but not pain-related behaviour, was blocked by co-administration of the cannabinoid receptor 1 (CB1) antagonist/inverse agonist rimonabant. Tissue levels of the endocannabinoids N-arachidonoylethanolamide (anandamide, AEA) and 2-AG were similar in the vHip of fear-conditioned rats receiving formalin injection and the vHip of fear-conditioned rats receiving saline injection. However, the levels of AEA and 2-AG were significantly lower in the contralateral ventrolateral periaqueductal grey of formalin-treated fear-conditioned rats than in that of their saline-treated counterparts. These data suggest that 2-AG-CB1 receptor signalling in the vHip has an anti-aversive effect, and that this effect is abolished in the presence of a persistent pain state.

Impaired endocannabinoid signalling in the rostral ventromedial medulla underpins genotype-dependent hyper-responsivity to noxious stimuli.

Pain is both a sensory and an emotional experience, and is subject to modulation by a number of factors including genetic background modulating stress/affect. The Wistar-Kyoto (WKY) rat exhibits a stress-hyper-responsive and depressive-like phenotype and increased sensitivity to noxious stimuli, compared with other rat strains. Here, we show that this genotype-dependent hyperalgesia is associated with impaired pain-related mobilisation of endocannabinoids and transcription of their synthesising enzymes in the rostral ventromedial medulla (RVM). Pharmacological blockade of the Cannabinoid1 (CB1) receptor potentiates the hyperalgesia in WKY rats, whereas inhibition of the endocannabinoid catabolising enzyme, fatty acid amide hydrolase, attenuates the hyperalgesia. The latter effect is mediated by CB1 receptors in the RVM. Together, these behavioural, neurochemical, and molecular data indicate that impaired endocannabinoid signalling in the RVM underpins hyper-responsivity to noxious stimuli in a genetic background prone to heightened stress/affect.

Cdc7-dependent and -independent phosphorylation of Claspin in the induction of the DNA replication checkpoint.

Claspin is a critical mediator protein in the DNA replication checkpoint, responsible for ATR-dependent activation of the effector kinase Chk1. Cdc7, an essential kinase required for the initiation of DNA replication, can also interact with and phosphorylate Claspin. In this study we use small-molecule inhibitors of Cdc7 kinase to further understand the relationship between Cdc7, Claspin and Chk1 activation. We demonstrate that inhibition of Cdc7 kinase delays HU-induced phosphorylation of Chk1 but does not affect the maintenance of the replication checkpoint once it is established. We find that while chromatin association of Claspin is not affected by Cdc7 inhibition, Claspin phosphorylation is attenuated following HU treatment, which may be responsible for the altered kinetics of HU-induced Chk1 phosphorylation. We demonstrate that Claspin is an in vitro substrate of Cdc7 kinase, and using mass-spectrometry, we identify multiple phosphorylation sites that help to define a Cdc7 phosphorylation motif. Finally, we show that the interaction between Claspin and Cdc7 is not dependent on Cdc7 kinase activity, but Claspin interaction with the DNA helicase subunit Mcm2 is lost upon Cdc7 inhibition. We propose Cdc7-dependent phosphorylation regulates critical protein-protein interactions and modulates Claspin's function in the DNA replication checkpoint.

Evidence for a role of GABAergic and glutamatergic signalling in the basolateral amygdala in endocannabinoid-mediated fear-conditioned analgesia in rats.

The basolateral amygdala (BLA) is a key substrate facilitating the expression of fear-conditioned analgesia (FCA). However, the neurochemical mechanisms in the BLA which mediate this potent suppression of pain responding during fear remain unknown. The present study investigated the role of cannabinoid1 (CB1) receptors and interactions with GABAergic (GABAA receptor) and glutamatergic (metabotropic glutamate receptor type 5; mGluR5) signalling in the BLA in formalin-evoked nociceptive behaviour and FCA in rats. Reexposure to a context previously paired with foot shock significantly reduced formalin-evoked nociceptive behaviour. Systemic or intra-BLA microinjection of the CB1 receptor antagonist/inverse agonist AM251 prevented this expression of FCA, while injection of AM251 into the central nucleus of the amygdala did not. The suppression of FCA by systemic AM251 administration was partially attenuated by intra-BLA administration of either the GABAA receptor antagonist bicuculline or the mGluR5 antagonist 2-methyl-6-(phenylethynyl) pyridine, (MPEP). Bilateral microinjection of MPEP, but not bicuculline, alone into the BLA enhanced formalin-evoked nociceptive behaviour. Postmortem analyses revealed that FCA was associated with a significant increase in tissue levels of anandamide in the BLA side contralateral to intraplantar formalin injection. In addition, fear-conditioned rats exhibited a robust formalin-induced increase in levels of 2-arachidonyl glycerol and N-palmitoylethanolamide in the ipsilateral and contralateral BLA, respectively. These data suggest that CB1 receptors in the BLA facilitate the expression of FCA, through a mechanism which is likely to involve the modulation of GABAergic and glutamatergic signalling.

Cross validation of liquid chromatography-mass spectrometry and lectin array for monitoring glycosylation in fed-batch glycoprotein production.

Glycosylation analysis of recombinant glycoproteins is of importance for the biopharmaceutical industry and the production of glycoprotein pharmaceuticals. A commercially available lectin array technology was evaluated for its ability to present a reproducible fingerprint of a recombinant CTLY4-IgG fusion glycoprotein expressed in large scale CHO-cell fermentation. The glycosylation prediction from the array was compared to traditional negative mode capillary LC-MS of released oligosaccharides. It was shown that both methods provide data that allow samples to be distinguished by their glycosylation pattern. This included information about sialylation, the presence of reducing terminal galactose ß1-, terminal N-acetylglucosamine ß1-, and antennary distribution. With both methods it was found that a general trend of increased sialylation was associated with an increase of the antenna and reduced amount of terminal galactose ß1-, while N-acetylglucosamine ß1- was less affected. LC-MS, but not the lectin array, provided valuable information about the sialic acid isoforms present, including N-acetylneuraminic acid, N-glycolylneuraminic acid and their O-acetylated versions. Detected small amounts of high-mannose structures by LC-MS correlated with the detection of the same epitope by the lectin array.

Negative ion CID fragmentation of O-linked oligosaccharide aldoses--charge induced and charge remote fragmentation.

Collision induced dissociation (CID) fragmentation was compared between reducing and reduced sulfated, sialylated, and neutral O-linked oligosaccharides. It was found that fragmentation of the [M - H](-) ions of aldoses with acidic residues gave unique Z-fragmentation of the reducing end GalNAc containing the acidic C-6 branch, where the entire C-3 branch was lost. This fragmentation pathway, which is not seen in the alditols, showed that the process involved charge remote fragmentation catalyzed by a reducing end acidic anomeric proton. With structures containing sialic acid on both the C-3 and C-6 branch, the [M - H](-) ions were dominated by the loss of sialic acid. This fragmentation pathway was also pronounced in the [M - 2H](2-) ions revealing both the C-6 Z-fragment plus its complementary C-3 C-fragment in addition to glycosidic and cross ring fragmentation. This generation of the Z/C-fragment pairs from GalNAc showed that the charges were not participating in their generation. Fragmentation of neutral aldoses showed pronounced Z-fragmentation believed to be generated by proton migration from the C-6 branch to the negatively charged GalNAc residue followed by charge remote fragmentation similar to the acidic oligosaccharides. In addition, A-type fragments generated by charge induced fragmentation of neutral oligosaccharides were observed when the charge migrated from C-1 of the GalNAc to the GlcNAc residue followed by rearrangement to accommodate the (0,2)A-fragmentation. LC-MS also showed that O-linked aldoses existed as interchangeable α/ß pyranose anomers, in addition to a third isomer (25% of the total free aldose) believed to be the furanose form.

A role for the ventral hippocampal endocannabinoid system in fear-conditioned analgesia and fear responding in the presence of nociceptive tone in rats.

The endogenous cannabinoid (endocannabinoid) system plays an important role in fear-conditioned analgesia (FCA) and expression and extinction of conditioned fear. The hippocampus has an established role in both pain and conditioned fear and is a substrate for endocannabinoid activity. This study aimed to investigate the role of the endocannabinoid system in the ventral hippocampus (vHip) in FCA and in fear responding in the presence of nociceptive tone. Fear-conditioned rats displayed significantly increased freezing and 22-kHz ultrasonic vocalisation and a reduction in formalin-evoked nociceptive behaviour (ie, FCA) upon re-exposure to a context previously paired with footshock. Tissue levels of the endocannabinoids, anandamide, and 2-arachidonoylglycerol, as well as the fatty acid amide, palmitoylethanolamide, were significantly higher in the vHip of fear-conditioned rats compared with non-fear-conditioned controls. URB597 (inhibitor of fatty acid amide hydrolase [FAAH]), administered bilaterally into the vHip, significantly enhanced FCA during the entire trial and increased fear responding in formalin-treated rats early in the trial. The URB597-induced enhancement of FCA was blocked by intra-vHip administration of the cannabinoid(1) (CB(1)) receptor antagonist/inverse agonist rimonabant. Intra-vHip rimonabant alone had no effect on the expression of FCA, and URB597 did not significantly alter formalin-evoked nociceptive behaviour in non-fear-conditioned rats. These data suggest an important role for the endocannabinoid system in the vHip in FCA, whereby levels of 2-arachidonoylglycerol and the FAAH substrates palmitoylethanolamide and anandamide are increased in rats expressing FCA, and pharmacological inhibition of FAAH in the vHip enhances this form of endogenous analgesia via a CB(1) receptor-dependent mechanism.

DNA mediated chromatin pull-down for the study of chromatin replication.

Chromatin replication involves duplicating DNA while maintaining epigenetic information. These processes are critical for genome stability and for preserving cell-type identity. Here we describe a simple experimental approach that allows chromatin to be captured and its content analysed after in vivo replication and labeling of DNA by cellular DNA polymerases. We show that this technique is highly specific and that proteins bound to the replicated DNA can be analyzed by both immunological techniques and large scale mass spectrometry. As proof of concept we have used this novel procedure to begin investigating the relationship between chromatin protein composition and the temporal programme of DNA replication in human cells. It is expected that this technique will become a widely used tool to address how chromatin proteins assemble onto newly replicated DNA after passage of a replication fork and how chromatin maturation is coupled to DNA synthesis.

Nimesulide 90 mg orally twice daily does not influence postoperative morphine requirements after major chest surgery.

BACKGROUND: Cyclooxygenase 2 inhibition has proven analgesic efficacy in a variety of surgical procedures. We postulated that perioperative cyclooxygenase 2 inhibition significantly reduces postoperative morphine requirements after major thoracic surgery and investigated the site of this potential analgesic effect. METHODS: Ninety-two patients participated in this single-center, double-blind, randomized, placebo-controlled, parallel-group trial. Patients between the ages of 18 and 80 yr undergoing a thoracotomy or median sternotomy were randomized to receive either nimesulide or placebo in combination with a standard analgesic regimen perioperatively. Nimesulide was administered orally the evening before surgery and at 12-h intervals for 5 days postoperatively. The primary efficacy variables were morphine consumption and pain scores for the first 48 h postoperatively. The secondary efficacy variable was the effect of nimesulide on cyclooxygenase activity in cerebrospinal fluid (CSF). RESULTS: Pain scores at rest or with movement, and total morphine consumption for the first 48 h postoperatively, were not statistically different between the groups. The mean difference in total morphine consumption up to 48 h postoperatively between the nimesulide and placebo group was a 9.0 mg reduction (95% CI: -28.9 to 10.9 mg) (P = 0.37). Adjusted mean (se) CSF 6-keto-PGF1alpha (6-keto-PGF1alpha) concentrations increased by 54.7 (25.7) pg/mL from preoperatively to Day + 2 postoperatively in the placebo group, whereas adjusted mean (se) CSF 6-keto-PGF1alpha concentration decreased by 0.6 pg/mL (18.2 pg/mL) in the nimesulide group. These changes were not statistically different between the groups (P = 0.095). CONCLUSION: Nimesulide, at a dose of 90 mg twice daily in combination with a standard analgesic regimen, does not influence pain scores, morphine requirements, or CSF prostaglandin levels after major thoracic surgery.

Profound resolution of early atherosclerosis with conjugated linoleic acid.

Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of linoleic acid and has been shown to suppress the development of atherosclerosis in experimental models. However, the mechanism involved is unclear although it is believed it may act as a cyclooxygenase inhibitor or as an agonist of the nuclear receptors, peroxisome proliferator activated receptors (PPARs). In this study, we examined the effect of cis-9,trans-11:trans-10,cis-12-CLA (80:20 blend) on the regression of pre-established atherosclerosis. ApoE(-/-) mice fed a 1% cholesterol diet were randomized at 8 weeks to continue receiving the diet supplemented with 1% control saturated fat or 1% CLA blend for a further 8 weeks. CLA supplementation did not simply prevent progression but induced almost complete resolution of atherosclerosis. Although CLA inhibited platelet deposition, as detected by staining of platelet glycoprotein alpha11b beta111a, it did not inhibit COX-mediated generation of prostaglandins in this model. However, PPARalpha and PPARgamma expression was increased in the aorta of the CLA-treated animals. This was coincident with decreased macrophage accumulation and decreased expression of the macrophage scavenger receptor CD36 and increased apoptosis in the aorta in vivo. CLA induces the resolution of atherosclerosis by negatively regulating the expression of pro-inflammatory genes and inducing apoptosis in the atherosclerotic lesion.