Question: Tirasemtiv has no effect on fast-twitch muscle.

Title: Activation of fast skeletal muscle troponin as a potential therapeutic approach for treating neuromuscular diseases Content: Limited neural input results in muscle weakness in neuromuscular disease because of a reduction in the density of muscle innervation, the rate of neuromuscular junction activation or the efficiency of synaptic transmission. We developed a small-molecule fast-skeletal-troponin activator, CK-2017357, as a means to increase muscle strength by amplifying the response of muscle when neural input is otherwise diminished secondary to neuromuscular disease. Binding selectively to the fast-skeletal-troponin complex, CK-2017357 slows the rate of calcium release from troponin C and sensitizes muscle to calcium. As a consequence, the force-calcium relationship of muscle fibers shifts leftwards, as does the force-frequency relationship of a nerve-muscle pair, so that CK-2017357 increases the production of muscle force in situ at sub-maximal nerve stimulation rates. Notably, we show that sensitization of the fast-skeletal-troponin complex to calcium improves muscle force and grip strength immediately after administration of single doses of CK-2017357 in a model of the neuromuscular disease myasthenia gravis. Troponin activation may provide a new therapeutic approach to improve physical activity in diseases where neuromuscular function is compromised.

Title: Hydrogen sulfide interacts with nitric oxide in the heart: possible involvement of nitroxyl. Content: AIMS The present study aims to investigate the interaction between nitric oxide (NO) and hydrogen sulfide (H(2)S), the two important gaseous mediators in rat hearts. METHODS AND RESULTS Intracellular calcium in isolated cardiomyocytes was measured with a spectrofluorometric method using Fura-2. Myocyte contractility was measured with a video edge system. NaHS (50 µM, an H(2)S donor) had no significant effect on the resting calcium level, electrically induced (EI) calcium transients, and cell contractility in ventricular myocytes. Stimulating endogenous NO production with l-arginine or exogenous application of NO donors [sodium nitroprusside (SNP) and 2-(N,N-diethylamino)-diazenolate-2-oxide] decreased myocyte twitch amplitudes accompanied by slower velocities of both cell contraction and relaxation. Surprisingly, NaHS reversed the negative inotropic and lusitropic effects of the above three NO-increasing agents. In addition, the mixture of SNP + NaHS increased, whereas SNP alone decreased, the resting calcium level and the amplitudes of EI calcium transients. Angeli's salt, a nitroxyl anion (HNO) donor, mimicked the effect of SNP + NaHS on calcium handling and myocyte contractility. Three thiols, N-acetyl-cysteine, l-cysteine, and glutathione, abolished the effects of HNO and SNP + NaHS on myocyte contraction. Neither Rp-cAMP [a protein kinase A (PKA) inhibitor] nor Rp-cGMP [a protein kinase G (PKG) inhibitor] affected the effects of SNP + NaHS, suggesting a cAMP/PKA- or cGMP/PKG-independent mechanism. CONCLUSION H(2)S may interact with NO to form a thiol sensitive molecule (probably HNO) which produces positive inotropic and lusitropic effects. Our findings may shed light on the interaction of NO and H(2)S and provide new clues to treat cardiovascular diseases.

Title: The Combination of Exercise and Respiratory Training Improves Respiratory Muscle Function in Pulmonary Hypertension Content: Increased dyspnea and reduced exercise capacity in pulmonary arterial hypertension (PAH) can be partly attributed to impaired respiratory muscle function. This prospective study was designed to assess the impact of exercise and respiratory training on respiratory muscle strength and 6-min walking distance (6MWD) in PAH patients. Patients with invasively confirmed PAH underwent 3 weeks of in-hospital exercise and respiratory training, which was continued at home for another 12 weeks. Medication remained constant during the study period. Blinded observers assessed efficacy parameters at baseline (I) and after 3 (II) and 15 weeks (III). Respiratory muscle function was assessed by twitch mouth pressure (TwPmo) during nonvolitional supramaximal magnetic phrenic nerve stimulation. Seven PAH patients (4 women; mean pulmonary artery pressure 45 ± 11 mmHg, median WHO functional class 3.1 ± 0.4, idiopathic/associated PAH n = 5/2) were included. The training program was feasible and well tolerated by all patients with excellent compliance. TwPmo was I: 0.86 ± 0.37 kPa, II: 1.04 ± 0.29 kPa, and III: 1.27 ± 0.44 kPa, respectively. 6MWD was I: 417 ± 51 m, II: 509 ± 39 m, and III: 498 ± 39 m, respectively. Both TwPmo (+0.41 ± 0.34 kPa, +56 ± 39 %) and 6MWD (+81 ± 30 m, +20 ± 9 %) increased significantly in the period between baseline and the final assessment (pairwise comparison: p = 0.012/<0.001; RM-ANOVA considering I, II, III: p = 0.037/<0.001). Exercise and respiratory training as an adjunct to medical therapy may be effective in patients with PAH to improve respiratory muscle strength and exercise capacity. Future, randomized, controlled trials should be carried out to further investigate these findings.

Title: Effects of Thiazolidinediones on Stroke Recovery: A Case-Matched Controlled Study Content: Anti-inflammatory therapy decreases infarct size and enhances stroke recovery. Thiazolidinedione peroxisome proliferator-activated receptor (PPAR)gamma agonists have potent anti-inflammatory and insulin-sensitizing anti-diabetic actions. Thirty stroke patients with type 2 diabetes admitted for acute inpatient stroke rehabilitation receiving pioglitazone or rosiglitazone were matched for age, sex, initial FIMTM score and interval post-stroke with 30 stroke patients with type 2 diabetes not receiving thiazolidinediones. Relevant outcome variables were compared for both groups. The thiazolidinedione treated group showed significantly greater mean improvement in FIMTM score compared to control group (25.6 ± 10.2 SD vs. 19.8 ± 10.5, respectively, P = 0.015). There was no significant difference in length of rehabilitation hospital stay (24.2 ± 7.6 vs. 25.1 ± 7.4 days, P = 0.657) or final discharge destination (home/institution, 19/11 versus 17/13, P = 0.792). Use of thiazolidinediones was associated with enhanced functional recovery in stroke patients with type 2 diabetes.

Title: Addition of a Gastrointestinal Microbiome Modulator to Metformin Improves Metformin Tolerance and Fasting Glucose Levels. Content: BACKGROUND Adverse effects of metformin are primarily related to gastrointestinal (GI) intolerance that could limit titration to an efficacious dose or cause discontinuation of the medication. Because some metformin side effects may be attributable to shifts in the GI microbiome, we tested whether a GI microbiome modulator (GIMM) used in combination with metformin would ameliorate the GI symptoms. METHODS A 2-period crossover study design was used with 2 treatment sequences, either placebo in period 1 followed by GIMM in period 2 or vice versa. Study periods lasted for 2 weeks, with a 2-week washout period between. During the first week, type 2 diabetes patients (T2D) who experienced metformin GI intolerance took 500 mg metformin along with their assigned NM504 (GIMM) or placebo treatment with breakfast and with dinner. In the second week, the 10 subjects took 500 mg metformin (t.i.d.), with GIMM or placebo consumed with the first and third daily metformin doses. Subjects were permitted to discontinue metformin dosing if it became intolerable. RESULTS The combination of metformin and GIMM treatment produced a significantly better tolerance score to metformin than the placebo combination (6.78 ± 0.65 [mean ± SEM] versus 4.45 ± 0.69, P = .0006). Mean fasting glucose levels were significantly (P < .02) lower with the metformin-GIMM combination (121.3 ± 7.8 mg/dl) than with metformin-placebo (151.9 ± 7.8 mg/dl). CONCLUSION Combining a GI microbiome modulator with metformin might allow the greater use of metformin in T2D patients and improve treatment of the disease.

Title: The different biological effects of telomestatin and TMPyP4 can be attributed to their selectivity for interaction with intramolecular or intermolecular G-quadruplex structures. Content: Demonstration of the existence of G-quadruplex structures in telomeres of Stylonychia macronuclei and in the promoter of c-myc in human cells has validated these secondary DNA structures as potential targets for drug design. The next important issue is the selectivity of G-quadruplex-interactive agents for the different types of G-quadruplex structures. In this study, we have taken an important step in associating specific biological effects of these drugs with selective interaction with either intermolecular or intramolecular G-quadruplex structures formed in telomeres. Telomestatin is a natural product isolated from Streptomyces anulatus 3533-SV4 and has been shown to be a very potent telomerase inhibitor through its G-quadruplex interaction. We have demonstrated that telomestatin interacts preferentially with intramolecular versus intermolecular G-quadruplex structures and also has a 70-fold selectivity for intramolecular G-quadruplex structures over duplex DNA. Telomestatin is able to stabilize G-quadruplex structures that are formed from duplex human telomeric DNA as well as from single-stranded DNA. Importantly, telomestatin stabilizes these G-quadruplex structures in the absence of monovalent cations, which is a unique characteristic among G-quadruplex-interactive compounds. At noncytotoxic concentrations, telomestatin suppresses the proliferation of telomerase-positive cells within several weeks. In contrast, TMPyP4, a compound that preferentially facilitates the formation of intermolecular G-quadruplex structures, suppresses the proliferation of alternative lengthening of telomeres (ALT)-positive cells as well as telomerase-positive cells. We have also demonstrated that TMPyP4 induces anaphase bridges in sea urchin embryos, whereas telomestatin did not have this effect, leading us to conclude that the selectivity of telomestatin for intramolecular G-quadruplex structures and TMPyP4 for intermolecular G-quadruplex structures is important in mediating different biological effects: stabilization of intramolecular G-quadruplex structures produces telomerase inhibition and accelerated telomere shortening, whereas facilitation of the formation of intermolecular G-quadruplex structures induces the formation of anaphase bridges.

Title: Topoisomerase II plays an essential role as a swivelase in the late stage of SV40 chromosome replication in vitro. Content: The effects of topoisomerases I and II on the replication of SV40 DNA were examined using an in vitro replication system of purified proteins that constitutes the monopolymerase system. In the presence of the two topoisomerases, two distinct nascent DNAs were formed. One product arising from the replication of the leading template strand was approximately half the size of the template DNA, whereas the other product derived from the lagging template strand consisted of short DNAs. These products were synthesized from both SV40 naked DNA and SV40 chromosomes. For the replication of SV40 naked DNA, either topoisomerase I or II maintained replication fork movement and supported complete leading strand synthesis. When SV40 chromosomes were replicated with the same proteins, reactions containing only topoisomerase I produced shorter leading strands. However, mature size DNA products accumulated in reactions supplemented with topoisomerase II, as well as in reactions containing only topoisomerase II. In the presence of crude extracts of HeLa cells, VP-16, a specific inhibitor of topoisomerase II, blocked elongation of the nascent DNA during the replication of SV40 chromosomes. These results indicate that topoisomerase II plays a crucial role as a swivelase in the late stage of SV40 chromosome replication in vitro.

Title: Effects of exercise training of 8 weeks and detraining on plasma levels of endothelium-derived factors, endothelin-1 and nitric oxide, in healthy young humans. Content: Vascular endothelial cells produce nitric oxide (NO), which is a potent vasodilator substance and has been proposed as having antiatherosclerotic property. Vascular endothelial cells also produce endothelin-1 (ET-1), which is a potent vasoconstrictor peptide and has potent proliferating activity on vascular smooth muscle cells. Therefore, ET-1 has been implicated in the progression of atheromatous vascular disease. Because exercise training has been reported to produce an alteration in the function of vascular endothelial cells in animals, we hypothesized that exercise training influences the production of NO and ET-1 in humans. The purpose of the present study was to examine whether chronic exercise could influence the plasma levels of NO (measured as the stable end product of NO, i.e., nitrite/nitrate [NOx]) and ET-1 in humans. Eight healthy young subjects (20.3 +/- 0.5 yr old) participated in the study and exercised by cycling on a leg ergometer (70% VO2max for 1 hour, 3-4 days/week) for 8 weeks. Venous plasma concentrations of NOx and ET-1 were measured before and after (immediately before the end of 8-week exercise training) the exercise training, and also after the 4th and 8th week after the cessation of training. The VO2max significantly increased after exercise training. After the exercise training, the plasma concentration of NOx significantly increased (30.69 +/- 3.20 vs. 48.64 +/- 8.16 micromol/L, p < 0.05), and the plasma concentration of ET-1 significantly decreased (1.65 +/- 0.14 vs. 1.23 +/- 0.12 pg/mL, p < 0.05). The increase in NOx level and the decrease in ET-1 level lasted to the 4th week after the cessation of exercise training and these levels (levels of NOx and ET-1) returned to the basal levels (the levels before the exercise training) in the 8th week after the cessation of exercise training. There was a significant negative correlation between plasma NOx concentration and plasma ET-1 concentration. The present study suggests that chronic exercise causes an increase in production of NO and a decrease in production of ET-1 in humans, which may produce beneficial effects (i.e., vasodilative and antiatherosclerotic) on the cardiovascular system.

Title: Acute exercise activates nuclear factor (NF)-kappaB signaling pathway in rat skeletal muscle. Content: Two studies were performed to investigate the effects of an acute bout of physical exercise on the nuclear protein kappaB (NF-kappaB) signaling pathway in rat skeletal muscle. In Study 1, a group of rats (n=6) was run on the treadmill at 25 m/min, 5% grade, for 1 h or until exhaustion (Ex), and compared with a second group (n=6) injected with two doses of pyrrolidine dithiocarbamate (PDTC, 100 mg/kg, i.p.) 24 and 1 h prior to the acute exercise bout. Three additional groups of rats (n=6) were injected with either 8 mg/kg (i.p.) of lipopolysaccharide (LPS), 1 mmol/kg (i.p.) t-butylhydroperoxide (tBHP), or saline (C) and killed at resting condition. Ex rats showed higher levels of NF-kappaB binding and P50 protein content in muscle nuclear extracts compared with C rats. Cytosolic IkappaBalpha and IkappaB kinase (IKK) contents were decreased, whereas phospho-IkappaBalpha and phospho-IKK contents were increased, comparing Ex vs. C. The exercise-induced activation of NF-kappaB signaling cascade was partially abolished by PDTC treatment. LPS, but not tBHP, treatment mimicked and exaggerated the effects observed in Ex rats. In Study 2, the time course of exercise-induced NF-kappaB activation was examined. Highest levels of NF-kappaB binding were observed at 2 h postexercise. Decreased cytosolic IkappaBalpha and increased phosphor-IkappaBalpha content were found 0-1 h postexercise whereas P65 reached peak levels at 2-4 h. These data suggest that the NF-kappaB signaling pathway can be activated in a redox-sensitive manner during muscular contraction, presumably due to increased oxidant production. The cascade of intracellular events may be the overture to elevated gene expression of manganese superoxide dismutase reported earlier (Pfluegers Arch. 442, 426-434, 2001).

Title: Ticagrelor vs. clopidogrel in patients with acute coronary syndromes and diabetes: a substudy from the PLATelet inhibition and patient Outcomes (PLATO) trial Content: AIMS patients with diabetes mellitus (DM) have high platelet reactivity and are at increased risk of ischaemic events and bleeding post-acute coronary syndromes (ACS). In the PLATelet inhibition and patient Outcomes (PLATO) trial, ticagrelor reduced the primary composite endpoint of cardiovascular death, myocardial infarction, or stroke, but with similar rates of major bleeding compared with clopidogrel. We aimed to investigate the outcome with ticagrelor vs. clopidogrel in patients with DM or poor glycaemic control. METHODS AND RESULTS we analysed patients with pre-existing DM (n = 4662), including 1036 patients on insulin, those without DM (n = 13 951), and subgroups based on admission levels of haemoglobin A1c (HbA1c; n = 15 150). In patients with DM, the reduction in the primary composite endpoint (HR: 0.88, 95% CI: 0.76-1.03), all-cause mortality (HR: 0.82, 95% CI: 0.66-1.01), and stent thrombosis (HR: 0.65, 95% CI: 0.36-1.17) with no increase in major bleeding (HR: 0.95, 95% CI: 0.81-1.12) with ticagrelor was consistent with the overall cohort and without significant diabetes status-by-treatment interactions. There was no heterogeneity between patients with or without ongoing insulin treatment. ticagrelor reduced the primary endpoint, all-cause mortality, and stent thrombosis in patients with HbA1c above the median (HR: 0.80, 95% CI: 0.70-0.91; HR: 0.78, 95% CI: 0.65-0.93; and HR: 0.62, 95% CI: 0.39-1.00, respectively) with similar bleeding rates (HR: 0.98, 95% CI: 0.86-1.12). CONCLUSION ticagrelor, when compared with clopidogrel, reduced ischaemic events in ACS patients irrespective of diabetic status and glycaemic control, without an increase in major bleeding events.

Title: Angiotensin II Evokes Angiogenic Signals within Skeletal Muscle through Co-ordinated Effects on Skeletal Myocytes and Endothelial Cells Content: Skeletal muscle overload induces the expression of angiogenic factors such as vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-2, leading to new capillary growth. We found that the overload-induced increase in angiogenesis, as well as increases in VEGF, MMP-2 and MT1-MMP transcripts were abrogated in muscle VEGF KO mice, highlighting the critical role of myocyte-derived VEGF in controlling this process. The upstream mediators that contribute to overload-induced expression of VEGF have yet to be ascertained. We found that muscle overload increased angiotensinogen expression, a precursor of angiotensin (Ang) II, and that Ang II signaling played an important role in basal VEGF production in C2C12 cells. Furthermore, matrix-bound VEGF released from myoblasts induced the activation of endothelial cells, as evidenced by elevated endothelial cell phospho-p38 levels. We also found that exogenous Ang II elevates VEGF expression, as well as MMP-2 transcript levels in C2C12 myotubes. Interestingly, these responses also were observed in skeletal muscle endothelial cells in response to Ang II treatment, indicating that these cells also can respond directly to the stimulus. The involvement of Ang II in muscle overload-induced angiogenesis was assessed. We found that blockade of AT1R-dependent Ang II signaling using losartan did not attenuate capillary growth. Surprisingly, increased levels of VEGF protein were detected in overloaded muscle from losartan-treated rats. Similarly, we observed elevated VEGF production in cultured endothelial cells treated with losartan alone or in combination with Ang II. These studies conclusively establish the requirement for muscle derived VEGF in overload-induced angiogenesis and highlight a role for Ang II in basal VEGF production in skeletal muscle. However, while Ang II signaling is activated following overload and plays a role in muscle VEGF production, inhibition of this pathway is not sufficient to halt overload-induced angiogenesis, indicating that AT1-independent signals maintain VEGF production in losartan-treated muscle.

Title: Pharmacological blockage of fibro/adipogenic progenitor expansion and suppression of regenerative fibrogenesis is associated with impaired skeletal muscle regeneration. Content: Acute skeletal muscle injury triggers an expansion of fibro/adipogenic progenitors (FAPs) and a transient stage of fibrogenesis characterized by extracellular matrix deposition. While the perpetuation of such phase can lead to permanent tissue scarring, the consequences of its suppression remain to be studied. Using a model of acute muscle damage we were able to determine that pharmacological inhibition of FAP expansion by Nilotinib, a tyrosine kinase inhibitor with potent antifibrotic activity, exerts a detrimental effect on myogenesis during regeneration. We found that Nilotinib inhibits the damage-induced expansion of satellite cells in vivo, but it does not affect in vitro proliferation, suggesting a non cell-autonomous effect. Nilotinib impairs regenerative fibrogenesis by preventing the injury-triggered expansion and differentiation of resident CD45(-):CD31(-):α7integrin(-):Sca1(+) mesenchymal FAPs. Our data support the notion that the expansion of FAPs and transient fibrogenesis observed during regeneration play an important trophic role toward tissue-specific stem cells.

Title: Wedelolactone exhibits anti-fibrotic effects on human hepatic stellate cell line LX-2. Content: Wedelolactone is a major coumarin of Eclipta prostrata, which is used for preventing liver damage. However the effects of wedelolactone on hepatic fibrosis remained unexplored. The purpose of this study was to demonstrate the anti-fibrotic effects of wedelolactone on activated human hepatic stellate cell (HSC) line LX-2 and the possible underlying mechanisms by means of MTT assay, Hoechst staining, as well as real-time quantitative PCR and western blot. The results showed that wedelolactone reduced the cellular viability of LX-2 in a time and dose-dependent manner. After treatment of wedelolactone, the expressions of collagen I and α-smooth muscle actin, two biomarkers of LX-2 activation, were remarkably decreased. The apoptosis of LX-2 cells was induced by wedelolactone accompanied with the decreasing expression of anti-apoptotic Bcl-2 and increasing expression of pro-apoptotic Bax. In addition, phosphorylated status of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was up-regulated, but not in p38. Moreover, wedelolactone significantly repressed the level of phosphorylated inhibitor of nuclear factor κB (IκB) and p65 in nucleus in spite of tumor necrosis factor-α stimulation. In conclusion, wedelolactone could significantly inhibit the activation of LX-2 cells, the underlying mechanisms of which included inducing Bcl-2 family involved apoptosis, up-regulating phosphorylated status of ERK and JNK expressions, and inhibiting nuclear factor-κB (NF-κB) mediated activity. Wedelolactone might present as a useful tool for the prevention and treatment of hepatic fibrosis.

Title: The selectivity of beta-adrenoceptor antagonists on isoprenaline-induced changes in heart rate, blood pressure, soleus muscle contractility and airways function in anaesthetized cats. Content: The beta-adrenoceptor antagonist of propranolol, metoprolol, atenolol and butoxamine in anaesthetized cats has been measured and compared with the activity of four synthetic phenylethanolamine derivatives. The effects of isoprenaline on four parameters in the anaesthetized cat: heart rate, blood pressure, soleus muscle contractility and airway reactance, were measured and the modification of the isoprenaline dose-response relation by each of the antagonist drugs assessed. Parallel shifts in log dose-response curves for isoprenaline were caused by propranolol for all parameters, by metoprolol and atenolol for each parameter except blood pressure, and butoxamine for each except soleus muscle and heart rate. Selectivity of action of the antagonists between different organs was measured by comparing DR10 values, computed from isoprenaline dose-ratios. Propranolol was the most potent antagonist and showed slight selectivity of action on soleus muscle compared with heart. Atenolol and metoprolol were approximately equipotent and were cardioselective at low doses only. Butoxamine was the least potent antagonist and possessed non-beta-adrenoceptor effects on the parameters measured. Each of the new compounds, 4'-bromo-2'-methoxy-N-isopropyl phenylethanolamine, the 4'-chloro- and 4'-methyl analogues, and 4'-methoxy-N-t-butyl phenylethanolamine, was a potent antagonist but did not exhibit any selectivity of action. The results suggest no clear separation of beta-adrenoceptors into beta 1- and beta 2-subclasses in organs of the cat. There is no apparent separation of beta-adrenoceptor-mediated effects on skeletal muscle and airways.

Title: Quercetin attenuates vascular calcification by inhibiting oxidative stress and mitochondrial fission. Content: Vascular calcification is a strong independent predictor of increased cardiovascular morbidity and mortality and has a high prevalence among patients with chronic kidney disease. The present study investigated the effects of quercetin on vascular calcification caused by oxidative stress and abnormal mitochondrial dynamics both in vitro and in vivo. Calcifying vascular smooth muscle cells (VSMCs) treated with inorganic phosphate (Pi) exhibited mitochondrial dysfunction, as demonstrated by decreased mitochondrial potential and ATP production. Disruption of mitochondrial structural integrity was also observed in a rat model of adenine-induced aortic calcification. Increased production of reactive oxygen species, enhanced expression and phosphorylation of Drp1, and excessive mitochondrial fragmentation were also observed in Pi-treated VSMCs. These effects were accompanied by mitochondria-dependent apoptotic events, including release of cytochrome c from the mitochondria into the cytosol and subsequent activation of caspase-3. Quercetin was shown to block Pi-induced apoptosis and calcification of VSMCs by inhibiting oxidative stress and decreasing mitochondrial fission by inhibiting the expression and phosphorylation of Drp1. Quercetin also significantly ameliorated adenine-induced aortic calcification in rats. In summary, our findings suggest that quercetin attenuates calcification by reducing apoptosis of VSMCs by blocking oxidative stress and inhibiting mitochondrial fission.

Title: Periostin Deficiency Increases Bone Damage and Impairs Injury Response to Fatigue Loading in Adult Mice Content: Bone damage removal and callus formation in response to fatigue loading are essential to prevent fractures. Periostin (Postn) is a matricellular protein that mediates adaptive response of cortical bone to loading. Whether and how periostin influences damage and the injury response to fatigue remains unknown. We investigated the skeletal response of Postn(-/-) and Postn(+/+) mice after fatigue stimulus by axial compression of their tibia. In Postn(+/+) mice, cracks number and surface (CsNb, CsS) increased 1h after fatigue, with a decrease in strength compared to non-fatigued tibia. At 15 days, CsNb had started to decline, while CtTV and CtBV increased in fatigued vs non-fatigued tibia, reflecting a woven bone response that was present in 75% of the fatigued bones. Cortical porosity and remodelling also prominently increased in the fatigued tibia of Postn(+/+) mice. At 30 days, paralleling a continuous removal of cortical damage, strength of the fatigued tibia was similar to the non-fatigue tibia. In Postn(-/-) mice, cracks were detectable even in the absence of fatigue, while the amount of collagen crosslinks and tissue hardness was decreased compared to Postn(+/+). Fatigue significantly increased CsNb and CsS in Postn(-/-), but was not associated with changes in CtTV and CtBV, as only 16% of the fatigued bones formed some woven bone. Cortical porosity and remodelling did not increase either after fatigue in Postn(-/-), and the level of damage remained high even after 30 days. As a result, strength remained compromised in Postn(-/-) mice. Contrary to Postn(+/+), which osteocytic lacunae showed a change in the degree of anisotropy (DA) after fatigue, Postn(-/-) showed no DA change. Hence periostin appears to influence bone materials properties, damage accumulation and repair, including local modeling/remodeling processes in response to fatigue. These observations suggest that the level of periostin expression could influence the propensity to fatigue fractures.

Title: The relationship of the level of cyclic amp to differentiation in primary cultures of chick muscle cells. Content: Abstract The effect of increased levels of cAMP upon the differentiation of primary cultures of chick myo blasts has been investigated. 0.1 mM But 2 cAMP or 1 mM 3-isobutyl-1-methylxanthine was added to the cultures 24 h after plating and maintained throughout the 70 h period of culture examined. Both reagents were found to markedly delay the time of fusion of the myoblasts but had no observable effect upon the increase in activity of creatine phosphokinase. Morphological examination of the cells revealed no difference in the relative numbers of myoblasts and fibroblasts between the control, But 2 cAMP and 3-isobutyl-1-methylxanthine cultures, but the latter reagent appeared to cause some inhibition of cell proliferation.

Title: Wedelolactone induces growth of breast cancer cells by stimulation of estrogen receptor signalling. Content: Wedelolactone, a plant coumestan, was shown to act as anti-cancer agent for breast and prostate carcinomas in vitro and in vivo targeting multiple cellular proteins including androgen receptors, 5-lipoxygenase and topoisomerase IIα. It is cytotoxic to breast, prostate, pituitary and myeloma cancer cell lines in vitro at μM concentrations. In this study, however, a novel biological activity of nM dose of wedelolactone was demonstrated. Wedelolactone acts as agonist of estrogen receptors (ER) α and β as demonstrated by transactivation of estrogen response element (ERE) in cells transiently expressing either ERα or ERβ and by molecular docking of this coumestan into ligand binding pocket of both ERα and ERβ. In breast cancer cells, wedelolactone stimulates growth of estrogen receptor-positive cells, expression of estrogen-responsive genes and activates rapid non-genomic estrogen signalling. All these effects can be inhibited by pretreatment with pure ER antagonist ICI 182,780 and they are not observed in ER-negative breast cancer cells. We conclude that wedelolactone acts as phytoestrogen in breast cancer cells by stimulating ER genomic and non-genomic signalling pathways.

Title: Morin Exhibits Anti-Inflammatory Effects on IL-1β-Stimulated Human Osteoarthritis Chondrocytes by Activating the Nrf2 Signaling Pathway Content: Background/Aims: Osteoarthritis (OA) is a multifactorial disease that is associated with inflammation in joints. The purpose of the present study was to investigate the anti-inflammatory activity and mechanism of morin on human osteoarthritis chondrocytes stimulated by IL-1β. Methods: The levels of NO and PGE2 were measured by the Griess method and ELISA. The levels of MMP1, MMP3, and MMP13 were also measured by ELISA. Results: The results revealed that IL-1β significantly increased the production of NO, PGE2, MMP1, MMP3, and MMP13. Additionally, the increases were significantly attenuated by treatment with morin. Furthermore, IL-1β-induced NF-κB activation was suppressed by morin. In addition, the expression of Nrf2 and HO-1 were increased by morin and knockdown of Nrf2 could prevent the anti-inflammatory effects of morin. Conclusion: In conclusion, this study suggested that morin attenuated IL-1β-induced inflammation by activating the Nrf2 signaling pathway.

Title: Different response of human glioma tumor-initiating cells to epidermal growth factor receptor kinase inhibitors. Content: Because a subpopulation of cancer stem cells (tumor-initiating cells, TICs) is believed to be responsible for the development, progression, and recurrence of many tumors, we evaluated the in vitro sensitivity of human glioma TICs to epidermal growth factor receptor (EGFR) kinase inhibitors (erlotinib and gefitinib) and possible molecular determinants for their effects. Cells isolated from seven glioblastomas (GBM 1-7) and grown using neural stem cell permissive conditions were characterized for in vivo tumorigenicity, expression of tumor stem cell markers (CD133, nestin), and multilineage differentiation properties, confirming that these cultures are enriched in TICs. TIC cultures were challenged with increasing concentrations of erlotinib and gefitinib, and their survival was evaluated after 1-4 days. In most cases, a time- and concentration-dependent cell death was observed, although GBM 2 was completely insensitive to both drugs, and GBM 7 was responsive only to the highest concentrations tested. Using a radioligand binding assay, we show that all GBM TICs express EGFR. Erlotinib and gefitinib inhibited EGFR and ERK1/2 phosphorylation/activation in all GBMs, irrespective of the antiproliferative response observed. However, under basal conditions GBM 2 showed a high Akt phosphorylation that was completely insensitive to both drugs, whereas GBM 7 was completely insensitive to gefitinib, and Akt inactivation occurred only for the highest erlotinib concentration tested, showing a precise relationship with the antiproliferative effects of the drug. Interestingly, in GBM 2, phosphatase and tensin homolog expression was significantly down-regulated, possibly accounting for the insensitivity to the drugs. In conclusion, glioma TICs are responsive to anti-EGFR drugs, but phosphatase and tensin homolog expression and Akt inhibition seem to be necessary for such effect.

Title: Hydrogen sulphide regulates intracellular pH in vascular smooth muscle cells. Content: We investigated the role of hydrogen sulphide (H(2)S) in intracellular pH (pH(i)) regulation in vascular smooth muscle cells and its contribution on vasodilation. NaHS, a H(2)S donor, decreased pH(i) in a concentration-dependent manner ranging from 10 microM to 1mM. Neither inhibition of the Na(+)/H(+) exchanger with 5-(N-ethyl-N-isopropyl) amiloride, (EIPA, 10 microM), nor plasmalemmal Ca(2+)-ATPase with CdCl(2) (20nM) alters the effect of NaHS on pH(i). Blockade of the Cl(-)/HCO3- exchanger with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) significantly attenuated the pH(i) lowering effect of NaHS. Moreover, NaHS significantly increased the activity of Cl(-)/HCO3- exchanger when measured with NH(4)Cl prepulse method. DIDS attenuated the vasorelaxation induced by NaHS whereas EIPA and CdCl(2) did not cause any change. In conclusion, H(2)S induced intracellular acidification via activation of Cl(-)/HCO3- exchanger, which is, at least partially, responsible for H(2)S-mediated vasorelaxation.

Title: Role of common hypnotics on the phenotypic causes of obstructive sleep apnoea: paradoxical effects of zolpidem. Content: Hypnotics are contraindicated in obstructive sleep apnoea (OSA) because of concerns of pharyngeal muscle relaxation and delayed arousal worsening hypoxaemia. However, human data are lacking. This study aimed to determine the effects of three common hypnotics on the respiratory arousal threshold, genioglossus muscle responsiveness and upper airway collapsibility during sleep.21 individuals with and without OSA (18-65 years) completed 84 detailed sleep studies after receiving temazepam (10 mg), zolpidem (10 mg), zopiclone (7.5 mg) and placebo on four occasions in a randomised, double-blind, placebo-controlled, crossover trial (ACTRN12612001004853).The arousal threshold increased with zolpidem and zopiclone versus placebo (mean±sd -18.3±10 and -19.1±9 versus -14.6±7 cmH2O; p=0.02 and p<0.001) but not with temazepam (-16.8±9 cmH2O; p=0.17). Genioglossus muscle activity during stable non-REM sleep and responsiveness during airway narrowing was not different with temazepam and zopiclone versus placebo but, paradoxically, zolpidem increased median muscle responsiveness three-fold during airway narrowing (median -0.15 (interquartile range -1.01 to -0.04) versus -0.05 (-0.29 to -0.03)% maximum EMG per cmH2O epiglottic pressure; p=0.03). The upper airway critical closing pressure did not change with any of the hypnotics. These doses of common hypnotics have differential effects on the respiratory arousal threshold but do not reduce upper airway muscle activity or alter airway collapsibility during sleep. Rather, muscle activity increases during airway narrowing with zolpidem.

Title: Behavioral characterization of neuropeptide Y knockout mice. Content: An extensive behavioral characterization was conducted with mice lacking the gene for neuropeptide Y (NPY) including response to 24 and 48 h fast and challenge with small molecule antagonists of NPY receptors implicated in mediating the feeding effects of NPY (i.e., Y1 and Y5). In addition, wildtype (WT) and NPY knockout (KO) mice were tested in locomotor monitors, elevated plus maze, inhibitory avoidance, acoustic startle, prepulse inhibition, and hot plate assays. One of the major findings was that the NPY KO mice have a reduced food intake relative to WT controls in response to fasting. Also, based on data from the behavioral models, the NPY KO mice may have an anxiogenic-like phenotype, and appear to be hypoalgesic in the hot plate paradigm. The data from these studies provide further evidence of involvement of NPY in energy balance, anxiety, and possibly nociception.

Title: CNTF reverses obesity-induced insulin resistance by activating skeletal muscle AMPK Content: Ciliary neurotrophic factor (CNTF) induces weight loss and improves glucose tolerance in humans and rodents. CNTF is thought to act centrally by inducing hypothalamic neurogenesis to modulate food intake and peripherally by altering hepatic gene expression, in a manner similar to that of leptin. Here, we show that CNTF signals through the CNTFRα–IL-6R–gp130β receptor complex to increase fatty-acid oxidation and reduce insulin resistance in skeletal muscle by activating AMP-activated protein kinase (AMPK), independent of signaling through the brain. Thus, our findings further show that the antiobesogenic effects of CNTF in the periphery result from direct effects on skeletal muscle, and that these peripheral effects are not suppressed by diet-induced or genetic models of obesity, an essential requirement for the therapeutic treatment of obesity-related diseases.

Title: Exercise causes a tissue-specific change of NO production in the kidney and lung. Content: Nitric oxide (NO) is produced in the vascular endothelium and is a potent vasodilator substance that participates in the regulation of local vascular tone. Exercise causes peculiar changes in systemic and regional blood flow, i.e., an increase of systemic blood flow and a redistribution of local tissue blood flow, by which the blood flow is greatly increased in the working muscles, whereas it is decreased in some organs such as the kidney and intestine. Thus we hypothesized that exercise causes a tissue-specific change of NO production in some internal organs. We studied whether exercise affects expression of NO synthase (NOS) mRNA and protein, NOS activity, and tissue level of nitrite/nitrate (stable end products of NO) in the kidneys (in which blood flow during exercise is decreased) and lungs (in which blood flow during exercise is increased with the increase of cardiac output) of rat. Rats ran on a treadmill for 45 min at a speed of 25 m/min. Immediately after this exercise, kidneys and lungs were quickly removed. Control rats remained at rest during this 45-min period. Expression of endothelial NOS (eNOS) mRNA in the kidneys was markedly lower in exercise rats than in control rats, whereas that in the lungs was significantly higher in exercise rats than in control rats. Western blot analysis confirmed down- and upregulation of eNOS protein in the kidney and lung, respectively, after exercise. On the other hand, neither expression of neuronal NOS (nNOS) mRNA and nNOS protein nor inducible NOS (iNOS) mRNA and iNOS protein in the kidneys and lungs differed between exercise and control rats. NOS activity in the kidney was significantly lower in exercise rats than in control rats, whereas that in the lung was significantly higher in exercise rats than in control rats. On the other hand, the iNOS activity in the kidneys and lungs did not differ between exercise rats and control rats. Tissue nitrite/nitrate level in the kidneys was markedly lower in exercise rats, whereas that in the lungs was significantly higher in exercise rats. The present results show that production of NO is markedly and tissue-specifically changed in the kidney and lung by exercise.

Title: High-Threshold Mechanosensitive Ion Channels Blocked by a Novel Conopeptide Mediate Pressure-Evoked Pain Content: Little is known about the molecular basis of somatosensory mechanotransduction in mammals. We screened a library of peptide toxins for effects on mechanically activated currents in cultured dorsal root ganglion neurons. One conopeptide analogue, termed NMB-1 for noxious mechanosensation blocker 1, selectively inhibits (IC50 1 µM) sustained mechanically activated currents in a subset of sensory neurons. Biotinylated NMB-1 retains activity and binds selectively to peripherin-positive nociceptive sensory neurons. The selectivity of NMB-1 was confirmed by the fact that it has no inhibitory effects on voltage-gated sodium and calcium channels, or ligand-gated channels such as acid-sensing ion channels or TRPA1 channels. Conversely, the tarantula toxin, GsMTx-4, which inhibits stretch-activated ion channels, had no effects on mechanically activated currents in sensory neurons. In behavioral assays, NMB-1 inhibits responses only to high intensity, painful mechanical stimulation and has no effects on low intensity mechanical stimulation or thermosensation. Unexpectedly, NMB-1 was found to also be an inhibitor of rapid FM1-43 loading (a measure of mechanotransduction) in cochlear hair cells. These data demonstrate that pharmacologically distinct channels respond to distinct types of mechanical stimuli and suggest that mechanically activated sustained currents underlie noxious mechanosensation. NMB-1 thus provides a novel diagnostic tool for the molecular definition of channels involved in hearing and pressure-evoked pain.

Title: Epigenetic Therapy Ties MYC Depletion to Reversing Immune Evasion and Treating Lung Cancer Content: Combining DNA-demethylating agents (DNA methyltransferase inhibitors [DNMTis]) with histone deacetylase inhibitors (HDACis) holds promise for enhancing cancer immune therapy. Herein, pharmacologic and isoform specificity of HDACis are investigated to guide their addition to a DNMTi, thus devising a new, low-dose, sequential regimen that imparts a robust anti-tumor effect for non-small-cell lung cancer (NSCLC). Using in-vitro-treated NSCLC cell lines, we elucidate an interferon α/β-based transcriptional program with accompanying upregulation of antigen presentation machinery, mediated in part through double-stranded RNA (dsRNA) induction. This is accompanied by suppression of MYC signaling and an increase in the T cell chemoattractant CCL5. Use of this combination treatment schema in mouse models of NSCLC reverses tumor immune evasion and modulates T cell exhaustion state towards memory and effector T cell phenotypes. Key correlative science metrics emerge for an upcoming clinical trial, testing enhancement of immune checkpoint therapy for NSCLC.

Title: Fibrillar Collagen Inhibits Arterial Smooth Muscle Proliferation through Regulation of Cdk2 Inhibitors Content: Arterial smooth muscle cells (SMCs) are arrested in the G1 phase of the cell cycle on polymerized type I collagen fibrils, while monomer collagen supports SMC proliferation. Cyclin E-associated kinase and cyclin-dependent kinase 2 (cdk2) phosphorylation are inhibited on polymerized collagen, and levels of the cdk2 inhibitors p27Kip1 and p21Cip1/Waf1 are increased compared with SMCs on monomer collagen. p27Kip1 associates with the cyclin E-cdk2-p21Cip1/Waf1 complex in SMCs on polymerized collagen. Monovalent blocking antibodies to alpha2 integrins, integrins that mediate adhesion to both forms of collagen, mimic these effects on monomer collagen. Furthermore, polymerized collagen rapidly suppresses p70 S6 kinase, a possible regulator of p27Kip1. Thus, fibrillar collagen specifically regulates early integrin signaling that may lead to up-regulation of cdk2 inhibitors and inhibition of SMC proliferation.

Title: Peroxisome proliferator-activated receptor ligand bezafibrate for prevention of type 2 diabetes mellitus in patients with coronary artery disease. Content: BACKGROUND Recent studies have shown that type 2 diabetes is preventable by both lifestyle interventions and medications that influence primary glucose metabolism. Whether pharmacological interventions that influence primary lipid metabolism can also delay development of type 2 diabetes is unknown. The goal of this study was to evaluate the effect of the peroxisome proliferator-activated receptor ligand bezafibrate on the progression of impaired fasting glucose phase to type 2 diabetes in patients with coronary artery disease over a 6.2-year follow-up period. METHODS AND RESULTS The study sample comprised 303 nondiabetic patients 42 to 74 years of age with a fasting blood glucose level of 110 to 125 mg/dL (6.1 to 6.9 mmol/L). The patients received either 400 mg bezafibrate retard (156 patients) or placebo (147 patients) once a day. No patients were using statins, and use of ACE inhibitors, which also reduce diabetes incidence, was relatively low. During follow-up, development of new-onset diabetes was recorded in 146 patients: in 80 (54.4%) from the placebo group and 66 (42.3%) from the bezafibrate group (P=0.04). The mean time until onset of new diabetes was significantly delayed in patients on bezafibrate compared with patients on placebo: 4.6+/-2.3 versus 3.8+/-2.6 years (P=0.004). Multivariate analysis identified bezafibrate treatment as an independent predictor of reduced risk of new diabetes development (hazard ratio, 0.70; 95% CI, 0.49 to 0.99). Other significant variables associated with future overt type 2 diabetes in patients with impaired fasting glucose were total cholesterol level (hazard ratio, 1.22; 95% CI 1.0 to 1.51) and body mass index (hazard ratio, 1.10; 95% CI, 1.05 to 1.16). CONCLUSIONS Bezafibrate reduces the incidence and delays the onset of type 2 diabetes in patients with impaired fasting glucose. Whether the combination of bezafibrate with other recommended drugs for secondary prevention (statins and ACE inhibitors) would be as efficacious as suggested by our results remains to be determined.

Title: Fasting Activation of AgRP Neurons Requires NMDA Receptors and Involves Spinogenesis and Increased Excitatory Tone Content: AgRP neuron activity drives feeding and weight gain whereas that of nearby POMC neurons does the opposite. However, the role of excitatory glutamatergic input in controlling these neurons is unknown. To address this question, we generated mice lacking NMDA receptors (NMDARs) on either AgRP or POMC neurons. Deletion of NMDARs from AgRP neurons markedly reduced weight, body fat and food intake whereas deletion from POMC neurons had no effect. Activation of AgRP neurons by fasting, as assessed by c-Fos, Agrp and Npy mRNA expression, AMPA receptor-mediated EPSCs, depolarization and firing rates, required NMDARs. Furthermore, AgRP but not POMC neurons have dendritic spines and increased glutamatergic input onto AgRP neurons caused by fasting was paralleled by an increase in spines, suggesting fasting induced synaptogenesis and spinogenesis. Thus glutamatergic synaptic transmission and its modulation by NMDARs play key roles in controlling AgRP neurons and determining the cellular and behavioral response to fasting.

Title: Effects of Electro-Acupuncture Therapy on Post-Stroke Depression in Patients with Different Degrees of Motor Function Impairments: a Pilot Study Content: [Purpose] The present study examined whether electro-acupuncture therapy reduces post-stroke depression (PSD) and whether motor function impairments interact with the effects of the therapy. [Subjects] Twenty-eight PSD patients were assessed and assigned to either a good or poor motor function group depending on their motor grade. [Methods] The Beck Depression Inventory (BDI), Hamilton Depression Rating Scale (HDRS) and Manual Muscle Test (MMT) were administered at the screening and initial phases of the study, and at the 4th, 8th, 12th and 16th week of the daily electro-acupuncture treatment. [Results] The electro-acupuncture treatment reduced PSD (as assessed by BDI and HDRS) of the patients. In particular, the depression of the good motor function group was significantly more reduced than that of the poor motor function group. The degree of motor function impairment did not change throughout the study in either group. [Conclusion] The results of the present study demonstrate that electro-acupuncture therapy can improve PSD, and that the treatment effect varies depending on the degree of motor function impairment.

Title: Peroxisome proliferator-activated receptor gamma agonists for the Prevention of Adverse events following percutaneous coronary Revascularization--results of the PPAR study. Content: BACKGROUND Patients with metabolic syndrome are at increased risk for cardiovascular complications. We sought to determine whether peroxisome proliferator-activated receptor gamma agonists had any beneficial effect on patients with metabolic syndrome undergoing percutaneous coronary intervention (PCI). METHODS A total of 200 patients with metabolic syndrome undergoing PCI were randomized to rosiglitazone or placebo and followed for 1 year. Carotid intima-medial thickness (CIMT), inflammatory markers, lipid levels, brain natriuretic peptide, and clinical events were measured at baseline, 6 months, and 12 months. RESULTS There was no significant difference in CIMT between the 2 groups. There was no difference in the 12-month composite end point of death, myocardial infarction (MI), stroke, or any recurrent ischemia (31.4% vs 30.2%, P = .99). The rate of death, MI, or stroke at 12 months was numerically lower in the rosiglitazone group (11.9% vs 6.4%, P = .19). There was a trend toward a greater decrease over time in high-sensitivity C-reactive protein values compared with baseline in the group randomized to rosiglitazone versus placebo both at 6 months (-35.4% vs -15.8%, P = .059) and 12 months (-40.0% vs -20.9%, P = .089) and higher change in high-density lipoprotein (+15.5% vs +4.1%, P = .05) and lower triglycerides (-13.9% vs +14.9%, P = .004) in the rosiglitazone arm. There was a trend toward less new onset diabetes in the rosiglitazone group (0% vs 3.3%, P = .081) and no episodes of symptomatic hypoglycemia. There was no excess of new onset of clinical heart failure in the rosiglitazone group, nor was there a significant change in brain natriuretic peptide levels. CONCLUSIONS Patients with metabolic syndrome presenting for PCI are at increased risk for subsequent cardiovascular events. Rosiglitazone for 12 months did not appear to affect CIMT in this population, although it did have beneficial effects on high-sensitivity C-reactive protein, high-density lipoprotein, and triglycerides. Further study of peroxisome proliferator-activated receptor agonism in patients with metabolic syndrome undergoing PCI may be warranted.

Title: Transient delivery of modified mRNA encoding TERT rapidly extends telomeres in human cells. Content: Telomere extension has been proposed as a means to improve cell culture and tissue engineering and to treat disease. However, telomere extension by nonviral, nonintegrating methods remains inefficient. Here we report that delivery of modified mRNA encoding TERT to human fibroblasts and myoblasts increases telomerase activity transiently (24-48 h) and rapidly extends telomeres, after which telomeres resume shortening. Three successive transfections over a 4 d period extended telomeres up to 0.9 kb in a cell type-specific manner in fibroblasts and myoblasts and conferred an additional 28 ± 1.5 and 3.4 ± 0.4 population doublings (PDs), respectively. Proliferative capacity increased in a dose-dependent manner. The second and third transfections had less effect on proliferative capacity than the first, revealing a refractory period. However, the refractory period was transient as a later fourth transfection increased fibroblast proliferative capacity by an additional 15.2 ± 1.1 PDs, similar to the first transfection. Overall, these treatments led to an increase in absolute cell number of more than 10(12)-fold. Notably, unlike immortalized cells, all treated cell populations eventually stopped increasing in number and expressed senescence markers to the same extent as untreated cells. This rapid method of extending telomeres and increasing cell proliferative capacity without risk of insertional mutagenesis should have broad utility in disease modeling, drug screening, and regenerative medicine.

Title: Loss of fibronectin from the aged stem cell niche affects the regenerative capacity of skeletal muscle in mice Content: Age-related changes in the niche have long been postulated to impair the function of somatic stem cells. Here we demonstrate that the aged stem cell niche in skeletal muscle contains substantially reduced levels of fibronectin (FN), leading to detrimental consequences for the function and maintenance of muscle stem cells (MuSCs). Deletion of the gene encoding FN from young regenerating muscles replicates the aging phenotype and leads to a loss of MuSC numbers. By using an extracellular matrix (ECM) library screen and pathway profiling, we characterize FN as a preferred adhesion substrate for MuSCs and demonstrate that integrin-mediated signaling through focal adhesion kinase and the p38 mitogen-activated protein kinase pathway is strongly de-regulated in MuSCs from aged mice because of insufficient attachment to the niche. Reconstitution of FN levels in the aged niche remobilizes stem cells and restores youth-like muscle regeneration. Taken together, we identify the loss of stem cell adhesion to FN in the niche ECM as a previously unknown aging mechanism.

Title: Fresh meat and further processing characteristics of ham muscles from finishing pigs fed ractopamine hydrochloride. Content: Ractopamine hydrochloride (RAC) has consistently led to an advantage in carcass cutting yields of finishing pigs and remains a common feed additive in US finishing pig diets. Less is known about the effect of RAC on further processing characteristics. Some researchers have reported advantages in ultimate pH of the LM in pigs fed RAC. If a greater ultimate pH was also observed in hams, the increased pH could affect further processing characteristics and lead to better protein interaction and improved textural properties. The objective of this experiment was to determine if RAC-fed pigs yielded hams with a greater ultimate pH, and if so, whether or not that advantage improves textural properties and water retention of further processed hams. Two hundred hams from barrows and gilts fed RAC or control diets were selected based on HCW. Hams were fabricated into 5 separate pieces to determine cutting yields, and 6 muscles were evaluated for ultimate pH. Hams were processed to make cured and smoked hams. Ractopamine increased cutting yields of the whole ham (P < 0.0001), inside (P < 0.01), outside (P < 0.01), and knuckle (P < 0.01) when expressed as a percentage of chilled side weight. Ultimate pH of the rectus femoris, vastus lateralis, and semitendinosus were all 0.06 pH units greater (P < 0.05), the biceps femoris was 0.04 pH units greater (P = 0.02), and the semimembranosus and adductor muscles were 0.03 pH units greater in pigs fed 7.4 mg/kg of RAC when compared with control pigs. Cured hams from RAC-fed pigs were heavier at all stages of production. No differences were detected in binding strengths (P = 0.88) or protein fat-free values (P = 0.13) between RAC (9.06 kg and 20.37) and control hams (9.01 kg and 20.13). Ractopamine increased cutting yields, total weight of cured hams, and ultimate muscle pH. Ractopamine can be fed to pigs to achieve the desired growth characteristic advantages and cutting yields without affecting further processed ham characteristics.

Title: Lack of Skeletal Muscle IL-6 Affects Pyruvate Dehydrogenase Activity at Rest and during Prolonged Exercise Content: Pyruvate dehydrogenase (PDH) plays a key role in the regulation of skeletal muscle substrate utilization. IL-6 is produced in skeletal muscle during exercise in a duration dependent manner and has been reported to increase whole body fatty acid oxidation, muscle glucose uptake and decrease PDHa activity in skeletal muscle of fed mice. The aim of the present study was to examine whether muscle IL-6 contributes to exercise-induced PDH regulation in skeletal muscle. Skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice and floxed littermate controls (control) completed a single bout of treadmill exercise for 10, 60 or 120 min, with rested mice of each genotype serving as basal controls. The respiratory exchange ratio (RER) was overall higher (P<0.05) in IL-6 MKO than control mice during the 120 min of treadmill exercise, while RER decreased during exercise independent of genotype. AMPK and ACC phosphorylation also increased with exercise independent of genotype. PDHa activity was in control mice higher (P<0.05) at 10 and 60 min of exercise than at rest but remained unchanged in IL-6 MKO mice. In addition, PDHa activity was higher (P<0.05) in IL-6 MKO than control mice at rest and 60 min of exercise. Neither PDH phosphorylation nor acetylation could explain the genotype differences in PDHa activity. Together, this provides evidence that skeletal muscle IL-6 contributes to the regulation of PDH at rest and during prolonged exercise and suggests that muscle IL-6 normally dampens carbohydrate utilization during prolonged exercise via effects on PDH.

Title: The emerging role of skeletal muscle extracellular matrix remodelling in obesity and exercise. Content: Skeletal muscle extracellular matrix remodelling has been proposed as a new feature associated with obesity and metabolic dysfunction. Exercise training improves muscle function in obesity, which may be mediated by regulatory effects on the muscle extracellular matrix. This review examined available literature on skeletal muscle extracellular matrix remodelling during obesity and the effects of exercise. A non-systematic literature review was performed on PubMed of publications from 1970 to 2015. A total of 37 studies from humans and animals were retained. Studies reported overall increases in gene and protein expression of different types of collagen, growth factors and enzymatic regulators of the skeletal muscle extracellular matrix in obesity. Only two studies investigated the effects of exercise on skeletal muscle extracellular matrix during obesity, with both suggesting a regulatory effect of exercise. The effects of exercise on muscle extracellular matrix seem to be influenced by the duration and type of exercise training with variable effects from a single session compared with a longer duration of exercise. More studies are needed to elucidate the mechanisms behind skeletal muscle extracellular matrix remodelling during obesity and the effects of exercise.

Title: Exercise blood flow patterns within and among rat muscles after training. Content: This study was designed to determine the influence of a long-term, moderate-intensity treadmill training program on the distribution of blood flow within and among muscles of rats during exercise. One group (T) of male Sprague-Dawley rats trained for 1 h/day for 13-17 wk at 30 m/min on a motor-driven treadmill. A second group (UT) of rats was conditioned for 10 min/day for 4 wk at the same speed. Muscle succinate dehydrogenase activities were higher in T than UT rats indicating a significant training effect. Blood flows (BFs) in 32 hindlimb muscles or muscle parts and other selected organs were measured in the two groups with radiolabeled microspheres during preexercise and while the rats ran for 30 s, 5 min, or 15 min at 30 m/min on the treadmill. The data indicate 1) there were no differences in total hindlimb muscle BF between UT and T rats at any time; however, 2) T rats had higher preexercise heart rates and higher muscle BFs in the deep red extensor muscles, suggesting a greater anticipatory response to the impending exercise; 3) T rats demonstrated more rapid elevations in BF in the red extensor muscles at the commencement of exercise; 4) T rats had higher BFs in red extensor muscles during exercise, whereas UT rats had higher BFs in white muscles; and 5) T rats maintained higher BFs in the visceral organs during exercise. These findings demonstrate that exercise training results in changes in the distribution of BF within and among muscles and among organs during exercise. Specifically, data indicate the high-oxidative motor units that are primarily recruited in the muscles during the initial stages of moderate treadmill exercise receive higher blood flows in the trained rats; this presumably contributes to increased resistance to fatigue.

Title: Neuron-specific expression of CuZnSOD prevents the loss of muscle mass and function that occurs in homozygous CuZnSOD-knockout mice. Content: Deletion of copper-zinc superoxide dismutase (CuZnSOD) in Sod1(-/-) mice leads to accelerated loss of muscle mass and force during aging, but the losses do not occur with muscle-specific deletion of CuZnSOD. To determine the role of motor neurons in the muscle decline, we generated transgenic Sod1(-/-) mice in which CuZnSOD was expressed under control of the synapsin 1 promoter (SynTgSod1(-/-) mice). SynTgSod1(-/-) mice expressed CuZnSOD in brain, spinal cord, and peripheral nerve, but not in other tissues. Sciatic nerve CuZnSOD content in SynTgSod1(-/-) mice was ~20% that of control mice, but no reduction in muscle mass or isometric force was observed in SynTgSod1(-/-) mice compared with control animals, whereas muscles of age-matched Sod1(-/-) mice displayed 30-40% reductions in mass and force. In addition, increased oxidative damage and adaptations in stress responses observed in muscles of Sod1(-/-) mice were absent in SynTgSod1(-/-) mice, and degeneration of neuromuscular junction (NMJ) structure and function occurred in Sod1(-/-) mice but not in SynTgSod1(-/-) mice. Our data demonstrate that specific CuZnSOD expression in neurons is sufficient to preserve NMJ and skeletal muscle structure and function in Sod1(-/-) mice and suggest that redox homeostasis in motor neurons plays a key role in initiating sarcopenia during aging.

Title: Inhibition of apical sodium-dependent bile acid transporter as a novel treatment for diabetes. Content: Bile acids are recognized as metabolic modulators. The present study was aimed at evaluating the effects of a potent Asbt inhibitor (264W94), which blocks intestinal absorption of bile acids, on glucose homeostasis in Zucker Diabetic Fatty (ZDF) rats. Oral administration of 264W94 for two wk increased fecal bile acid concentrations and elevated non-fasting plasma total Glp-1. Treatment of 264W94 significantly decreased HbA1c and glucose, and prevented the drop of insulin levels typical of ZDF rats in a dose-dependent manner. An oral glucose tolerance test revealed up to two-fold increase in plasma total Glp-1 and three-fold increase in insulin in 264W94 treated ZDF rats at doses sufficient to achieve glycemic control. Tissue mRNA analysis indicated a decrease in farnesoid X receptor (Fxr) activation in small intestines and the liver but co-administration of a Fxr agonist (GW4064) did not attenuate 264W94 induced glucose lowering effects. In summary, our results demonstrate that inhibition of Asbt increases bile acids in the distal intestine, promotes Glp-1 release and may offer a new therapeutic strategy for type 2 diabetes mellitus.

Title: MicroRNA-29 induces cellular senescence in aging muscle through multiple signaling pathways Content: The mechanisms underlying the development of aging-induced muscle atrophy are unclear. By microRNA array and individual qPCR analyses, we found significant up-regulation of miR-29 in muscles of aged rodents vs. results in young. With aging, p85α, IGF-1 and B-myb muscle levels were lower while the expression of certain cell arrest proteins (p53, p16 and pRB) increased. When miR-29 was expressed in muscle progenitor cells (MPC), their proliferation was impaired while SA-βgal expression increased signifying the development of senescence. Impaired MPC proliferation resulted from interactions between miR-29 and the 3'-UTR of p85a, IGF-1 and B-myb, suppressing the translation of these mediators of myoblast proliferation. In vivo, electroporation of miR-29 into muscles of young mice suppressed the proliferation and increased levels of cellular arrest proteins, recapitulating aging-induced responses in muscle. A potential stimulus of miR-29 expression is Wnt-3a since we found that exogenous Wnt-3a stimulated miR-29 expression 2.7-fold in primary cultures of MPCs. Thus, aging-induced muscle senescence results from activation of miR-29 by Wnt-3a leading to suppressed expression of several signaling proteins (p85α, IGF-1 and B-myb) that act coordinately to impair the proliferation of MPCs contributing to muscle atrophy. The increase in miR-29 provides a potential mechanism for aging-induced sarcopenia.

Title: Consumption of isoflavone-rich soy protein does not alter homocysteine or markers of inflammation in postmenopausal women Content: Background/Objective:To investigate the effect of soy protein containing isoflavones on homocysteine (Hcy), C-reactive protein (CRP), soluble E-selectin (sE-selectin), soluble vascular adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1).Subject/Methods:In a randomized crossover design, 34 postmenopausal women consumed soy protein isolate (26±5 g protein containing 44±8 mg isoflavones per day) or milk protein isolate (26±5 g protein per day) for 6 weeks each. Fasting blood samples were collected at the end of each diet period and end points analyzed by enzyme-linked immunosorbent assay. Results:Concentrations of Hcy, CRP, sE-selectin, sVCAM-1 and sICAM-1 were not different between soy and milk diet treatments. Results did not differ by equol production status or by baseline lipid concentration. Adjustment for intake of folate and methionine did not alter the Hcy results. Conclusions:These data suggest that decreasing vascular inflammation and Hcy concentration are not likely mechanisms by which soy consumption reduces coronary heart disease risk.

Title: Noxa controls Mule-dependent Mcl-1 ubiquitination through the regulation of the Mcl-1/USP9X interaction. Content: The level of the Mcl-1 pro-survival protein is highly regulated, and the down-regulation of Mcl-1 expression favors the apoptotic process. Mcl-1 physically interacts with different BH3-only proteins; particularly, Noxa is involved in the modulation of Mcl-1 expression. In this study, we demonstrated that Noxa triggers the degradation of Mcl-1 at the mitochondria according to the exclusive location of Noxa at this compartment. The Noxa-induced degradation of Mcl-1 required the E3 ligase Mule, which is responsible for the polyubiquitination of Mcl-1. Because the USP9X deubiquitinase was recently demonstrated to be involved in Mcl-1 protein turnover by preventing its degradation through the removal of conjugated ubiquitin, we investigated whether Noxa affected the deubiquitination process. Interestingly, Noxa over-expression caused a decrease in the USP9X/Mcl-1 interaction associated with an increase in the Mcl-1 polyubiquitinated forms. Additionally, Noxa over-expression triggered an increase in the Mule/Mcl-1 interaction in parallel with the decrease in Mule/USP9X complex formation. Taken together, these modifications result in the degradation of Mcl-1 by the proteasome machinery. The implication of Noxa in the regulation of Mcl-1 proteasomal degradation adds complexity to this process, which is governed by multiple interactions.

Title: Temporal regulation of topoisomerase IV activity in E. coli. Content: We isolated a mutant allele of dnaX, encoding the tau and gamma subunits of the DNA polymerase III holoenzyme, that causes extreme cell filamentation but does not affect either cell growth or DNA replication. This phenotype results from a defect in daughter chromosome decatenation during rapid growth. In these cells, ParC, one subunit of topoisomerase IV, no longer associated with the replication factory, as occurs in wild-type cells, and was instead distributed uniformly on the nucleoid; the distribution of ParE, the other subunit of topoisomerase IV, was unaffected. In addition, the majority of topoisomerase IV activity in synchronized cell populations was restricted to late in the cell cycle, when replication was essentially complete. These observations suggest that topoisomerase IV activity in vivo might be dependent on release of ParC from the replication factory.

Title: The changes of antioxidant defense system caused by quercetin administration do not lead to DNA damage and apoptosis in the spleen and bone marrow cells of rats. Content: Quercetin may have the opposite effect, namely anti- as well as pro-oxidant. The aim of this study was to assess the results of quercetin anti- and/or pro-oxidant activity in the bone marrow and spleen cells of rats. The experimental rats were treated daily, with quercetin in a dose of 8 or 80mg/kg b.w. by gavage for 40 days. The intracellular redox state in cells were assessed by measuring the ferric ion reducing antioxidant power (FRAP) level and malonodialdehyde concentration. HO-1 mRNA expression was examined with real-time PCR. The extent of DNA damage was determined by the alkaline-labile comet assay. A potential pro-apoptotic quercetin action was determined using the FITC-Annexin V kit. The quercetin and isorhamnetin concentrations in serum were analyzed by HPLC-ECD. MDA concentration and FRAP values, were significantly decreased in the spleen and bone marrow cells of rats treated with quercetin, in a dose of 80mg/kg b.w. in comparison with the control rats; no significant changes were observed after quercetin was administered in a dose ten times as low. Treatment with quercetin dose-dependently upregulated the expression of HO-1 mRNA in the bone marrow cells. Quercetin administration to the rats did not induce either DNA damage or apoptosis in the examined cells. The results of our study prove that changes in the antioxidant state, caused by quercetin, do not lead to DNA damage or exert any pro-apoptotic activity in vivo.

Title: Suppression of intestinal neoplasia by deletion of Dnmt3b Content: Aberrant gene silencing accompanied by DNA methylation is associated with neoplastic progression in many tumors that also show global loss of DNA methylation. Using conditional inactivation of de novo methyltransferase Dnmt3b in Apc(Min/+) mice, we demonstrate that the loss of Dnmt3b has no impact on microadenoma formation, which is considered the earliest stage of intestinal tumor formation. Nevertheless, we observed a significant decrease in the formation of macroscopic colonic adenomas. Interestingly, many large adenomas showed regions with Dnmt3b inactivation, indicating that Dnmt3b is required for initial outgrowth of macroscopic adenomas but is not required for their maintenance. These results support a role for Dnmt3b in the transition stage between microadenoma formation and macroscopic colonic tumor growth and further suggest that Dnmt3b, and by extension de novo methylation, is not required for maintaining tumor growth after this transition stage has occurred.

Title: Comparison of pioglitazone vs glimepiride on progression of coronary atherosclerosis in patients with type 2 diabetes: the PERISCOPE randomized controlled trial. Content: CONTEXT No antidiabetic regimen has demonstrated the ability to reduce progression of coronary atherosclerosis. Commonly used oral glucose-lowering agents include sulfonylureas, which are insulin secretagogues, and thiazolidinediones, which are insulin sensitizers. OBJECTIVE To compare the effects of an insulin sensitizer, pioglitazone, with an insulin secretagogue, glimepiride, on the progression of coronary atherosclerosis in patients with type 2 diabetes. DESIGN, SETTING, AND PARTICIPANTS Double-blind, randomized, multicenter trial at 97 academic and community hospitals in North and South America (enrollment August 2003-March 2006) in 543 patients with coronary disease and type 2 diabetes. INTERVENTIONS A total of 543 patients underwent coronary intravascular ultrasonography and were randomized to receive glimepiride, 1 to 4 mg, or pioglitazone, 15 to 45 mg, for 18 months with titration to maximum dosage, if tolerated. Atherosclerosis progression was measured by repeat intravascular ultrasonography examination in 360 patients at study completion. MAIN OUTCOME MEASURE Change in percent atheroma volume (PAV) from baseline to study completion. RESULTS Least squares mean PAV increased 0.73% (95% CI, 0.33% to 1.12%) with glimepiride and decreased 0.16% (95% CI, -0.57% to 0.25%) with pioglitazone(P = .002). An alternative analysis imputing values for noncompleters based on baseline characteristics showed an increase in PAV of 0.64% (95% CI, 0.23% to 1.05%) for glimepiride and a decrease of 0.06% (-0.47% to 0.35%) for pioglitazone (between-group P = .02). Mean (SD) baseline HbA(1c) levels were 7.4% (1.0%) in both groups and declined during treatment an average 0.55% (95% CI, -0.68% to -0.42%) with pioglitazone and 0.36% (95% CI, -0.48% to -0.24%) with glimepiride (between-group P = .03). In the pioglitazone group, compared with glimepiride, high-density lipoprotein levels increased 5.7 mg/dL (95% CI, 4.4 to 7.0 mg/dL; 16.0%) vs 0.9 mg/dL (95% CI, -0.3 to 2.1 mg/dL; 4.1%), and median triglyceride levels decreased 16.3 mg/dL (95% CI, -27.7 to -11.0 mg/dL; 15.3%) vs an increase of 3.3 mg/dL (95% CI, -10.7 to 11.7 mg/dL; 0.6%) (P < .001 for both comparisons). Median fasting insulin levels decreased with pioglitazone and increased with glimepiride (P < .001). Hypoglycemia was more common in the glimepiride group and edema, fractures, and decreased hemoglobin levels occurred more frequently in the pioglitazone group. CONCLUSION In patients with type 2 diabetes and coronary artery disease, treatment with pioglitazone resulted in a significantly lower rate of progression of coronary atherosclerosis compared with glimepiride. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00225277.

Title: Predicting new molecular targets for known drugs Content: Although drugs are intended to be selective, at least some bind to several physiological targets, explaining side effects and efficacy. Because many drug-target combinations exist, it would be useful to explore possible interactions computationally. Here we compared 3,665 US Food and Drug Administration (FDA)-approved and investigational drugs against hundreds of targets, defining each target by its ligands. Chemical similarities between drugs and ligand sets predicted thousands of unanticipated associations. Thirty were tested experimentally, including the antagonism of the beta(1) receptor by the transporter inhibitor Prozac, the inhibition of the 5-hydroxytryptamine (5-HT) transporter by the ion channel drug Vadilex, and antagonism of the histamine H(4) receptor by the enzyme inhibitor Rescriptor. Overall, 23 new drug-target associations were confirmed, five of which were potent (<100 nM). The physiological relevance of one, the drug N,N-dimethyltryptamine (DMT) on serotonergic receptors, was confirmed in a knockout mouse. The chemical similarity approach is systematic and comprehensive, and may suggest side-effects and new indications for many drugs.

Title: Troponin concentrations for stratification of patients with acute coronary syndromes in relation to therapeutic efficacy of tirofiban. PRISM Study Investigators. Platelet Receptor Inhibition in Ischemic Syndrome Management. Content: BACKGROUND A major challenge for physicians is to identify patients with acute coronary syndromes who may benefit from treatment with glycoprotein-IIb/IIIa-receptor antagonists. We investigated whether troponin concentrations can be used to stratify patients for benefit from treatment with tirofiban. METHODS We enrolled 2222 patients of the Platelet Receptor Inhibition in Ischemic Syndrome Management study with coronary artery disease and who had had chest pain in the previous 24 h. All patients received aspirin and were randomly assigned treatment with tirofiban or heparin. We took baseline measurements of troponin I and troponin T. We recorded death, myocardial infarction, or recurrent ischaemia after 48 h infusion treatment and at 7 days and 30 days. FINDINGS 629 (28.3%) patients had troponin I concentrations higher than the diagnostic threshold of 1.0 microg/L and 644 (29.0%) troponin T concentrations higher than 0.1 microg/L. 30-day event rates (death, myocardial infarction) were 13.0% for troponin-I-positive patients compared with 4.9% for troponin-I-negative patients (p<0.0001), and 13.7% compared wth 3.5% for troponin T (p<0.001). At 30 days, in troponin-I-positive patients, tirofiban had lowered the risk of death (adjusted hazard ratio 0.25 [95% CI 0.09-0.68], p=0.004) and myocardial infarction (0.37 [0.16-0.84], p=0.01). This benefit was seen in medically managed patients (0.30 [0.10-0.84], p=0.004) and those undergoing revascularisation (0.37 [0.15-0.93] p=0.02) after 48 h infusion treatment. By contrast, no treatment effect was seen for troponin-I-negative patients. Similar benefits were seen for troponin-T-positive patients. INTERPRETATION Troponin I and troponin T reliably identified high-risk patients with acute coronary syndromes, managed medically and by revascularisation, who would benefit from tirofiban.

Title: Effects of PCSK9 Inhibition With Alirocumab on Lipoprotein Metabolism in Healthy Humans Content: BACKGROUND Alirocumab, a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 (PCSK9), lowers plasma low-density lipoprotein (LDL) cholesterol and apolipoprotein B100 (apoB). Although studies in mice and cells have identified increased hepatic LDL receptors as the basis for LDL lowering by PCSK9 inhibitors, there have been no human studies characterizing the effects of PCSK9 inhibitors on lipoprotein metabolism. In particular, it is not known whether inhibition of PCSK9 has any effects on very low-density lipoprotein or intermediate-density lipoprotein (IDL) metabolism. Inhibition of PCSK9 also results in reductions of plasma lipoprotein (a) levels. The regulation of plasma Lp(a) levels, including the role of LDL receptors in the clearance of Lp(a), is poorly defined, and no mechanistic studies of the Lp(a) lowering by alirocumab in humans have been published to date. METHODS Eighteen (10 F, 8 mol/L) participants completed a placebo-controlled, 2-period study. They received 2 doses of placebo, 2 weeks apart, followed by 5 doses of 150 mg of alirocumab, 2 weeks apart. At the end of each period, fractional clearance rates (FCRs) and production rates (PRs) of apoB and apo(a) were determined. In 10 participants, postprandial triglycerides and apoB48 levels were measured. RESULTS Alirocumab reduced ultracentrifugally isolated LDL-C by 55.1%, LDL-apoB by 56.3%, and plasma Lp(a) by 18.7%. The fall in LDL-apoB was caused by an 80.4% increase in LDL-apoB FCR and a 23.9% reduction in LDL-apoB PR. The latter was due to a 46.1% increase in IDL-apoB FCR coupled with a 27.2% decrease in conversion of IDL to LDL. The FCR of apo(a) tended to increase (24.6%) without any change in apo(a) PR. Alirocumab had no effects on FCRs or PRs of very low-density lipoproteins-apoB and very low-density lipoproteins triglycerides or on postprandial plasma triglycerides or apoB48 concentrations. CONCLUSIONS Alirocumab decreased LDL-C and LDL-apoB by increasing IDL- and LDL-apoB FCRs and decreasing LDL-apoB PR. These results are consistent with increases in LDL receptors available to clear IDL and LDL from blood during PCSK9 inhibition. The increase in apo(a) FCR during alirocumab treatment suggests that increased LDL receptors may also play a role in the reduction of plasma Lp(a). CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01959971.

Title: Chronic Exercise Increases Both Inducible and Endothelial Nitric Oxide Synthase Gene Expression in Endothelial Cells of Rat Aorta Content: Chronic exercise upregulates endothelial nitric oxide synthase (eNOS) gene expression. Whether the expression of inducible nitric oxide synthase (iNOS) is affected by exercise is unknown. We therefore investigated the effects of chronic exercise on iNOS and eNOS expression in isolated rat aortic endothelial and smooth muscle cells separately. Five-week-old male Wistar rats were randomly divided into control and exercise groups. After 10 weeks of running training, animals were sacrificed under ether anesthesia. The standard curve quantitative competitive reverse transcriptase-polymerase chain reaction method was used to quantify NOS mRNA expression in isolated endothelial/smooth muscle cells. To evaluate the functional role of iNOS, we examined phenylephrine-induced vascular responses with or without pretreatment with aminoguanidine. We found that (1) expression of iNOS and eNOS mRNA in endothelial cells was increased by chronic exercise and (2) chronic exercise blunted phenylephrine-induced vascular responses, probably by increasing NO release via iNOS. Our results show that chronic exercise increases both iNOS and eNOS gene expression in endothelium. These alterations may be partially responsible for the change in vascular response after exercise.

Title: Differential effects of charybdotoxin on the activity of retinal ganglion cells in the dark- and light-adapted mouse retina Content: Patch-clamp recordings were made from retinal ganglion cells in the mouse retina. Under dark adaptation, blockage of BK(Ca) channels increases the spontaneous excitatory postsynaptic currents (EPSCs) and light-evoked On-EPSCs, while it decreases the light-evoked Off inhibitory postsynaptic currents (IPSCs). However, under light adaptation it decreases the light-evoked On-EPSCs, the spontaneous IPSCs and the light-evoked On- and Off-IPSCs. Blockage of BK(Ca) channels significantly altered the outputs of RGCs by changing their light-evoked responses into a bursting pattern and increasing the light-evoked depolarization of the membrane potentials, while it did not significantly change the peak firing rates of light-evoked responses.

Title: Valvular interstitial cells suppress calcification of valvular endothelial cells. Content: BACKGROUND Calcific aortic valve disease (CAVD) is the most common heart valve disease in the Western world. We previously proposed that valvular endothelial cells (VECs) replenish injured adult valve leaflets via endothelial-to-mesenchymal transformation (EndMT); however, whether EndMT contributes to valvular calcification is unknown. We hypothesized that aortic VECs undergo osteogenic differentiation via an EndMT process that can be inhibited by valvular interstitial cells (VICs). APPROACH AND RESULTS VEC clones underwent TGF-β1-mediated EndMT, shown by significantly increased mRNA expression of the EndMT markers α-SMA (5.3 ± 1.2), MMP-2 (13.5 ± 0.6) and Slug (12 ± 2.1) (p < 0.05), (compared to unstimulated controls). To study the effects of VIC on VEC EndMT, clonal populations of VICs were derived from the same valve leaflets, placed in co-culture with VECs, and grown in control/TGF-β1 supplemented media. In the presence of VICs, EndMT was inhibited, shown by decreased mRNA expression of α-SMA (0.1 ± 0.5), MMP-2 (0.1 ± 0.1), and Slug (0.2 ± 0.2) (p < 0.05). When cultured in osteogenic media, VECs demonstrated osteogenic changes confirmed by increase in mRNA expression of osteocalcin (8.6 ± 1.3), osteopontin (3.7 ± 0.3), and Runx2 (5.5 ± 1.5). The VIC presence inhibited VEC osteogenesis, demonstrated by decreased expression of osteocalcin (0.4 ± 0.1) and osteopontin (0.2 ± 0.1) (p < 0.05). Time course analysis suggested that EndMT precedes osteogenesis, shown by an initial increase of α-SMA and MMP-2 (day 7), followed by an increase of osteopontin and osteocalcin (day 14). CONCLUSIONS The data indicate that EndMT may precede VEC osteogenesis. This study shows that VICs inhibit VEC EndMT and osteogenesis, indicating the importance of VEC-VIC interactions in valve homeostasis.

Title: Dnmt3a and Dnmt3b Associate with Enhancers to Regulate Human Epidermal Stem Cell Homeostasis. Content: The genome-wide localization and function of endogenous Dnmt3a and Dnmt3b in adult stem cells are unknown. Here, we show that in human epidermal stem cells, the two proteins bind in a histone H3K36me3-dependent manner to the most active enhancers and are required to produce their associated enhancer RNAs. Both proteins prefer super-enhancers associated to genes that either define the ectodermal lineage or establish the stem cell and differentiated states. However, Dnmt3a and Dnmt3b differ in their mechanisms of enhancer regulation: Dnmt3a associates with p63 to maintain high levels of DNA hydroxymethylation at the center of enhancers in a Tet2-dependent manner, whereas Dnmt3b promotes DNA methylation along the body of the enhancer. Depletion of either protein inactivates their target enhancers and profoundly affects epidermal stem cell function. Altogether, we reveal novel functions for Dnmt3a and Dnmt3b at enhancers that could contribute to their roles in disease and tumorigenesis.

Title: Gene expression in human skeletal muscle: alternative normalization method and effect of repeated biopsies Content: The reverse transcriptase-polymerase chain reaction (RT-PCR) method has lately become widely used to determine transcription and mRNA content in rodent and human muscle samples. However, the common use of endogenous controls for correcting for variance in cDNA between samples is not optimal. Specifically, we investigated (1) a new normalization method based on determining the cDNA content by the flourophores PicoGreen and OliGreen, (2) effect of repeated muscle biopsies on mRNA gene expression, and (3) the spatial heterogeneity in mRNA expression across the muscle. Standard curves using oligo standards revealed a high degree of sensitivity and linearity (2.5–45 ng; R 2>0.99) with OliGreen reagent, as was the case for OliGreen analyses with standard curves constructed from serial dilutions of representative RT samples (R 2 >0.99 for a ten times dilution range of a representative reversed transcribed (RT) sample). Likewise, PicoGreen reagent detected the RNA:DNA hybrid content in RT samples with great sensitivity. Standard curves constructed from both double-stranded lambda DNA (1–10 ng) and from serial dilutions of representative RT samples consistently resulted in linearity with R 2 >0.99. The present determination of cDNA content in reversed transcribed human skeletal muscle RNA samples by both PicoGreen and OliGreen analyses suggests that these fluorophores provide a potential alternative normalization procedure for human gene expression studies. In addition, the present study shows that multiple muscle biopsies obtained from the same muscle do not influence the mRNA response induced by an acute exercise bout for any of the genes examined.

Title: The Spemann Organizer Signal noggin Binds and Inactivates Bone Morphogenetic Protein 4 Content: Signals released by the Spemann organizer of the amphibian gastrula can directly induce neural tissue from ectoderm and can dorsalize ventral mesoderm to form muscle. The secreted polypeptide noggin mimics these activities and is expressed at the appropriate time and place to participate in the organizer signal. Neural induction and mesoderm dorsalization are antagonized by bone morphogenetic proteins (BMPs), which induce epidermis and ventral mesoderm instead. Here we report that noggin protein binds BMP4 with high affinity and can abolish BMP4 activity by blocking binding to cognate cell-surface receptors. These data suggest that noggin secreted by the organizer patterns the embryo by interrupting BMP signaling.

Title: Effect of losartan on ambulatory short-term blood pressure variability and cardiovascular remodeling in hypertensive patients on hemodialysis. Content: OBJECTIVE Previous studies have shown increases in ambulatory short-term blood pressure (BP) variability to be related to cardiovascular disease. In this study, we examined whether an angiotensin II type 1 receptor blocker losartan would improve ambulatory short-term BP variability in hypertensive patients on hemodialysis. METHODS Forty hypertensive patients on hemodialysis therapy were randomly assigned to the losartan treatment group (n=20) or the control treatment group (n=20). At baseline and 6 and 12 months after the treatment, 24-h ambulatory BP monitoring was performed. Echocardiography and measurements of brachial-ankle pulse wave velocity (baPWV) and biochemical parameters were also performed before and after therapy. RESULTS After 6- and 12-months of treatment, nighttime short-term BP variability, assessed on the basis of the coefficient of variation of ambulatory BP, was significantly decreased in the losartan group, but remained unchanged in the control group. Compared with the control group, losartan significantly decreased left ventricular mass index (LVMI), baPWV, and the plasma levels of brain natriuretic peptide and advanced glycation end products (AGE). Furthermore, multiple regression analysis showed significant correlations between changes in LVMI and changes in nighttime short-term BP variability, as well as between changes in LVMI and changes in the plasma levels of AGE. CONCLUSION These results suggest that losartan is beneficial for the suppression of pathological cardiovascular remodeling though its inhibitory effect on ambulatory short-term BP variability during nighttime.

Title: No effect of dietary fat on short-term weight gain in mice treated with atypical antipsychotic drugs Content: Rationale:Atypical antipsychotic drugs (AAD) induce significant weight gain in female C57BL/6J mice. The effect of dietary fat on weight gain and serum lipids in this model is unknown. Objectives: Test the hypothesis that the obesigenic effects of these drugs are greater in the presence of a high-fat diet. Methods:Female C57BL/6J mice were treated with atypical antipsychotics for 3 weeks and fed either a low-fat or high-fat diet (4.6 vs 15.6% fat by wt). Food intake (FI), body weight (BW), body composition, and serum lipids were measured during treatment with optimized doses of olanzapine, quetiapine, and risperidone. Energy intake (EI) and feed efficiency (FE) were calculated. Group differences in change were analyzed via repeated measures analysis of variance (ANOVA). Serum lipid concentrations, EI and FE were compared using two-way ANOVA.Results:AAD-treated mice gained significantly more weight than controls after 3 weeks (P<0.001). Treatment and diet had significant effects on FI and EI over time (P<0.001). AAD-treated mice had significantly higher FE than controls (P<0.05); however, there was no significant drug by diet interaction (P=0.65). Risperidone low-fat mice gained significantly more absolute fat mass than placebo low-fat mice (P<0.05). All treatment groups, except quetiapine low-fat and olanzapine high-fat, gained significantly more absolute lean mass than placebo controls (P<0.05). Cholesterol levels were significantly lower in quetiapine and risperidone than placebo (P<0.05). Risperidone low-fat mice had significantly higher triglyceride levels than placebo and risperidone high-fat mice (P<0.05).Conclusions:A high-fat diet does not increase AAD-induced BW gain in female mice during a 3-week treatment period.

Title: In vitro Anti-Tumor Effects of Statins on Head and Neck Squamous Cell Carcinoma: A Systematic Review Content: BACKGROUND Statins are commonly used against arteriosclerotic disease, but recent retrospective analyses have suggested that statins also prevent cancer. The aim of this systematic review is to verify the vitro anti-tumor effects of statins on head and neck squamous cell carcinoma. METHODS Studies were gathered by searching Cochrane, MEDLINE, EMBASE, LILACS, and PubMed, up until May 9, 2015, with no time or language restrictions. Only in vitro studies that discuss the effect of statins on head and neck carcinoma were selected. RESULTS Of 153 identified papers, 14 studies met the inclusion criteria. These studies demonstrated that statins had a significant effect on head and neck squamous cell carcinoma cell lines and influenced cell viability, cell cycle, cell death, and protein expression levels involved in pathways of carcinogenesis, which corroborates with the potential in vitro anti-tumor effects. It provides highlights about the biological mechanisms of statins used alone or associated with traditional therapy for cancer. CONCLUSIONS Though there are few studies on the topic, currently available evidence suggests that statins shows that preclinical experiments supports the potentiality of statin as an adjuvant agent in chemotherapy and/or radiotherapy approaches routinely used in the management of HNSCC and should undergo further clinical assessment.

Title: The effects of genetic and pharmacological blockade of the CB1 cannabinoid receptor on anxiety. Content: The aim of this study was to compare the effects of the genetic and pharmacological disruption of CB1 cannabinoid receptors on the elevated plus-maze test of anxiety. In the first experiment, the behaviour of CB1-knockout mice and wild-type mice was compared. In the second experiment, the cannabinoid antagonist SR141716A (0, 1, and 3 mg/kg) was administered to both CB1-knockout and wild type mice. Untreated CB1-knockout mice showed a reduced exploration of the open arms of the plus-maze apparatus, thus appearing more anxious than the wild-type animals, however no changes in locomotion were noticed. The vehicle-injected CB1-knockout mice from the second experiment also showed increased anxiety as compared with wild types. Surprisingly, the cannabinoid antagonist SR141716A reduced anxiety in both wild type and CB1 knockout mice. Locomotor behaviour was only marginally affected. Recent evidence suggests the existence of a novel cannabinoid receptor in the brain. It has also been shown that SR141716A binds to both the CB1 and the putative novel receptor. The data presented here supports these findings, as the cannabinoid receptor antagonist affected anxiety in both wild type and CB1-knockout mice. Tentatively, it may be suggested that the discrepancy between the effects of the genetic and pharmacological blockade of the CB1 receptor suggests that the novel receptor plays a role in anxiety.

Title: Nitrite protects against morbidity and mortality associated with TNF- or LPS-induced shock in a soluble guanylate cyclase–dependent manner Content: Nitrite (NO(2)(-)), previously viewed as a physiologically inert metabolite and biomarker of the endogenous vasodilator NO, was recently identified as an important biological NO reservoir in vasculature and tissues, where it contributes to hypoxic signaling, vasodilation, and cytoprotection after ischemia-reperfusion injury. Reduction of nitrite to NO may occur enzymatically at low pH and oxygen tension by deoxyhemoglobin, deoxymyoglobin, xanthine oxidase, mitochondrial complexes, or NO synthase (NOS). We show that nitrite treatment, in sharp contrast with the worsening effect of NOS inhibition, significantly attenuates hypothermia, mitochondrial damage, oxidative stress and dysfunction, tissue infarction, and mortality in a mouse shock model induced by a lethal tumor necrosis factor challenge. Mechanistically, nitrite-dependent protection was not associated with inhibition of mitochondrial complex I activity, as previously demonstrated for ischemia-reperfusion, but was largely abolished in mice deficient for the soluble guanylate cyclase (sGC) alpha1 subunit, one of the principal intracellular NO receptors and signal transducers in the cardiovasculature. Nitrite could also provide protection against toxicity induced by Gram-negative lipopolysaccharide, although higher doses were required. In conclusion, we show that nitrite can protect against toxicity in shock via sGC-dependent signaling, which may include hypoxic vasodilation necessary to maintain microcirculation and organ function, and cardioprotection.

Title: Pleiotropic effects of statins. Content: Statins are potent inhibitors of cholesterol biosynthesis. In clinical trials, statins are beneficial in the primary and secondary prevention of coronary heart disease. However, the overall benefits observed with statins appear to be greater than what might be expected from changes in lipid levels alone, suggesting effects beyond cholesterol lowering. Indeed, recent studies indicate that some of the cholesterol-independent or "pleiotropic" effects of statins involve improving endothelial function, enhancing the stability of atherosclerotic plaques, decreasing oxidative stress and inflammation, and inhibiting the thrombogenic response. Furthermore, statins have beneficial extrahepatic effects on the immune system, CNS, and bone. Many of these pleiotropic effects are mediated by inhibition of isoprenoids, which serve as lipid attachments for intracellular signaling molecules. In particular, inhibition of small GTP-binding proteins, Rho, Ras, and Rac, whose proper membrane localization and function are dependent on isoprenylation, may play an important role in mediating the pleiotropic effects of statins.

Title: PKD1 Inhibits AMPKα2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells. Content: AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser(485/491) of its α1/α2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser(485/491) phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser(485/491) phosphorylation, which was associated with a ∼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser(485/491) phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPKα2 at Ser(491) in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser(491) that plays a negative role in insulin signaling in muscle cells.

Title: Effect of alendronate on risk of fracture in women with low bone density but without vertebral fractures: results from the Fracture Intervention Trial. Content: CONTEXT Alendronate sodium reduces fracture risk in postmenopausal women who have vertebral fractures, but its effects on fracture risk have not been studied for women without vertebral fractures. OBJECTIVE To test the hypothesis that 4 years of alendronate would decrease the risk of clinical and vertebral fractures in women who have low bone mineral density (BMD) but no vertebral fractures. DESIGN Randomized, blinded, placebo-controlled trial. SETTING Eleven community-based clinical research centers. SUBJECTS Women aged 54 to 81 years with a femoral neck BMD of 0.68 g/cm2 or less (Hologic Inc, Waltham, Mass) but no vertebral fracture; 4432 were randomized to alendronate or placebo and 4272 (96%) completed outcome measurements at the final visit (an average of 4.2 years later). INTERVENTION All participants reporting calcium intakes of 1000 mg/d or less received a supplement containing 500 mg of calcium and 250 IU of cholecalciferol. Subjects were randomly assigned to either placebo or 5 mg/d of alendronate sodium for 2 years followed by 10 mg/d for the remainder of the trial. MAIN OUTCOME MEASURES Clinical fractures confirmed by x-ray reports, new vertebral deformities detected by morphometric measurements on radiographs, and BMD measured by dual x-ray absorptiometry. RESULTS Alendronate increased BMD at all sites studied (P<.001) and reduced clinical fractures from 312 in the placebo group to 272 in the intervention group, but not significantly so (14% reduction; relative hazard [RH], 0.86; 95% confidence interval [CI], 0.73-1.01). Alendronate reduced clinical fractures by 36% in women with baseline osteoporosis at the femoral neck (>2.5 SDs below the normal young adult mean; RH, 0.64; 95% CI, 0.50-0.82; treatment-control difference, 6.5%; number needed to treat [NNT], 15), but there was no significant reduction among those with higher BMD (RH, 1.08; 95% CI, 0.87-1.35). Alendronate decreased the risk of radiographic vertebral fractures by 44% overall (relative risk, 0.56; 95% CI, 0.39-0.80; treatment-control difference, 1.7%; NNT, 60). Alendronate did not increase the risk of gastrointestinal or other adverse effects. CONCLUSIONS In women with low BMD but without vertebral fractures, 4 years of alendronate safely increased BMD and decreased the risk of first vertebral deformity. Alendronate significantly reduced the risk of clinical fractures among women with osteoporosis but not among women with higher BMD.

Title: No net renal extraction of homocysteine in fasting humans. Content: BACKGROUND The pathophysiological mechanism of hyperhomocysteinemia in chronic renal failure in humans is unknown. The loss of a putative renal homocysteine extraction in chronic renal failure has been hypothesized as significant homocysteine uptake has been demonstrated in the normal rat kidney. We studied homocysteine extraction in the normal human kidney. METHODS We measured plasma total (free and protein-bound) and free homocysteine (tHcy and fHcy, respectively) in arterial and renal venous blood sampled from the aorta and right-side renal vein during cardiac catheterization in 20 fasting patients with normal renal function. Renal homocysteine extraction was calculated as the arteriovenous difference divided by the arterial levels times 100%. RESULTS No significant renal extraction was demonstrated either for tHcy: 0.9% (SD 5.8; 95% CI -1.8 to +3.6) or for fHcy: -0.2% (11.0; -5.4 to +4.9). CONCLUSIONS We conclude that no significant net renal uptake of homocysteine occurs in fasting humans with normal renal function. The loss of such uptake, therefore, cannot cause hyperhomocysteinemia in patients with renal failure.

Title: ACTA PSYCHIATRICA Content: OBJECTIVE We explored the subjective effects associated with olanzapine, risperidone and older antipsychotics. METHOD We conducted a content analysis of an Internet database of comments about prescribed medications. RESULTS We analysed 223 comments on risperidone, 170 on olanzapine and 46 relating to three older antipsychotics. The predominant subjective effects produced by all drugs consisted of sedation, cognitive impairment and emotional flattening or indifference. Connections appeared between these effects and Parkinsonian-like symptoms with the older drugs, sexual impairment with risperidone and metabolic effects with olanzapine. The experience of akathisia was frequently linked to suicidal thoughts. Some respondents described how the drugs' subjective effects helped to reduce symptoms of mania, psychosis and anxiety. CONCLUSION The generalisability of Internet data is uncertain. However, the data suggest that adverse subjective effects play a central role in the experience of taking antipsychotic drugs and may be related to the drugs' desired benefits.

Title: Do Montelukast Sodium and N-Acetylcysteine Have a Nephroprotective Effect on Unilateral Ureteral Obstruction? A Placebo Controlled Trial in a Rat Model. Content: PURPOSE We assessed the nephroprotective effects of montelukast sodium and N-acetylcysteine on secondary renal damage due to unilateral ureteral obstruction in a rat model. MATERIALS AND METHODS In this study 30 Wistar albino male rats were randomized into 3 groups, including placebo, N-acetylcysteine and montelukast sodium. Three rats served as the control group. The left ureter of the rats was sutured with 4-zero polyglactin sutures. Medications were given 3 days before obstruction and continued for 15 days. Dimercaptosuccinic acid renal scintigraphy was performed before obstruction and on day 15. Rats were sacrificed on day 15 and histopathological examinations were done. We biochemically assessed oxidative stress markers (myeloperoxidase and malondialdehyde), sulfhydryl and total nitrite for lipid peroxidation, oxidative protein damage and antioxidant levels, respectively. RESULTS On pathological examination inflammation and tubular epithelial damage in the N-acetylcysteine and montelukast sodium groups were less than in the placebo group (p <0.05). No difference was seen in normal kidneys. Myeloperoxidase, malondialdehyde and total nitrite levels in the N-acetylcysteine group, and myeloperoxidase and malondialdehyde levels in the montelukast sodium group were lower than in the placebo group (p <0.05). No statistical difference was seen in sulfhydryl levels (p >0.05) or among the N-acetylcysteine, montelukast sodium and placebo groups on scintigraphy (p >0.05). No pathological, chemical and scintigraphic differences were seen among the N-acetylcysteine, montelukast sodium and sham treated groups (p >0.05). CONCLUSIONS N-acetylcysteine and montelukast sodium have a protective effect against obstructive damage of the kidney. However, further investigations are needed.

Title: Epicatechin and a cocoa polyphenolic extract modulate gene expression in human Caco-2 cells. Content: We performed a functional genomic analysis to study the effect of epicatechin and polyphenolic cocoa extract in the human colon adenocarcinoma cell line Caco-2. The specific Human Hematology/Immunology cDNA arrays by Clontech, containing 406 genes in duplicate, were used. The differentially expressed genes were classified according to their level of expression, calculated as the ratio of the value obtained after each treatment relative to control cells, with a statistical significance of P < 0.05 (upregulated: ratio > 1.5; downregulated: ratio < 0.6). Treatment with epicatechin decreased the expression of 21 genes and upregulated 24 genes. Upon incubation with the cocoa polyphenolic extract, 24 genes were underexpressed and 28 were overexpressed. The changes in expression for ferritin heavy polypeptide 1 (FTH1), mitogen-activated protein kinase kinase 1 (MAPKK1), signal transducer and activator of transcription 1 (STAT1), and topoisomerase 1 upon incubation with epicatechin, and for myeloid leukemia factor 2 (MLF2), CCAAT/enhancer binding protein gamma (C/EBPG), MAPKK1, ATP-binding cassette, subfamily c member 1 (MRP1), STAT1, topoisomerase 1, and x-ray repair complementing defective repair 1 (XRCC1) upon incubation with the cocoa polyphenolic extract were validated by RT-PCR. Changes in the messenger RNA levels for MAPKK1, STAT1, MRP1, and topoisomerase 1 upon incubation with either epicatechin or cocoa extract were further confirmed at the protein level by Western blotting. The changes in the expression of STAT1, MAPKK1, MRP1, and FTH1 genes, which are involved in the cellular response to oxidative stress, are in agreement with the antioxidant properties of cocoa flavonoids. In addition, the changes in the expression of C/EBPG, topoisomerase 1, MLF2, and XRCC1 suggest novel mechanisms of action of flavonoids at the molecular level.

Title: The effect of N-acetylcysteine in the nucleus accumbens on neurotransmission and relapse to cocaine. Content: BACKGROUND Relapse to cocaine seeking has been linked with low glutamate in the nucleus accumbens core (NAcore) causing potentiation of synaptic glutamate transmission from prefrontal cortex (PFC) afferents. Systemic N-acetylcysteine (NAC) has been shown to restore glutamate homeostasis, reduce relapse to cocaine seeking, and depotentiate PFC-NAcore synapses. Here, we examine the effects of NAC applied directly to the NAcore on relapse and neurotransmission in PFC-NAcore synapses, as well as the involvement of the metabotropic glutamate receptors 2/3 (mGluR2/3) and 5 (mGluR5). METHODS Rats were trained to self-administer cocaine for 2 weeks and following extinction received either intra-accumbens NAC or systemic NAC 30 or 120 minutes, respectively, before inducing reinstatement with a conditioned cue or a combined cue and cocaine injection. We also recorded postsynaptic currents using in vitro whole cell recordings in acute slices and measured cystine and glutamate uptake in primary glial cultures. RESULTS NAC microinjection into the NAcore inhibited the reinstatement of cocaine seeking. In slices, a low concentration of NAC reduced the amplitude of evoked glutamatergic synaptic currents in the NAcore in an mGluR2/3-dependent manner, while high doses of NAC increased amplitude in an mGluR5-dependent manner. Both effects depended on NAC uptake through cysteine transporters and activity of the cysteine/glutamate exchanger. Finally, we showed that by blocking mGluR5 the inhibition of cocaine seeking by NAC was potentiated. CONCLUSIONS The effect of NAC on relapse to cocaine seeking depends on the balance between stimulating mGluR2/3 and mGluR5 in the NAcore, and the efficacy of NAC can be improved by simultaneously inhibiting mGluR5.

Title: Efficacy of Beta1 Integrin and EGFR Targeting in Sphere-Forming Human Head and Neck Cancer Cells Content: BACKGROUND Resistance to radiotherapy continues to be a limiting factor in the treatment of cancer including head and neck squamous cell carcinoma (HNSCC). Simultaneous targeting of β1 integrin and EGFR was shown to have a higher radiosensitizing potential than mono-targeting in the majority of tested HNSCC cancer models. As tumor-initiating cells (TIC) are thought to play a key role for therapy resistance and recurrence and can be enriched in sphere forming conditions, this study investigated the efficacy of β1 integrin/EGFR targeting without and in combination with X-ray irradiation on the behavior of sphere-forming cells (SFC). METHODS HNSCC cell lines (UTSCC15, UTSCC5, Cal33, SAS) were injected subcutaneously into nude mice for tumor up-take and plated for primary and secondary sphere formation under non-adhesive conditions which is thought to reflect the enrichment of SFC and their self-renewal capacity, respectively. Treatment was accomplished by inhibitory antibodies for β1 integrin (AIIB2) and EGFR (Cetuximab) as well as X-ray irradiation (2 - 6 Gy single doses). Further, flow cytometry for TIC marker expression and cell cycling as well as Western blotting for DNA repair protein expression and phosphorylation were employed. RESULTS We found higher primary and secondary sphere forming capacity of SAS cells relative to other HNSCC cell lines, which was in line with the tumor up-take rates of SAS versus UTSCC15 cells. AIIB2 and Cetuximab administration had minor cytotoxic and no radiosensitizing effects on SFC. Intriguingly, secondary SAS spheres, representing the fraction of surviving SFC upon passaging, showed greatly enhanced radiosensitivity compared to primary spheres. Intriguingly, neither AIIB2 nor Cetuximab significantly altered basal sphere forming capacity and radiosensitivity. While an increased accumulation of G0/G1 phase cells was observable in secondary SAS spheres, DNA double strand break repair indicated no difference on the basis of significantly enhanced ATM and Chk2 dephosphorylation upon irradiation. CONCLUSIONS In the HNSCC model, sphere-forming conditions select for cells, which are unsusceptible to both anti-β1 integrin and anti-EGFR inhibitory antibodies. With regard to primary and secondary sphere formation, our data suggest that both of these SFC fractions express distinct survival strategies independent from β1 integrin and EGFR and that future work is warranted to better understand SFC survival and enrichment before and after treatment to untangle the underlying mechanisms for identifying novel, druggable cancer targets in SFC.

Title: Once-a-day Concerta methylphenidate versus three-times-daily methylphenidate in laboratory and natural settings. Content: OBJECTIVE Methylphenidate (MPH), the most commonly prescribed drug for attention-deficit/hyperactivity disorder (ADHD), has a short half-life, which necessitates multiple daily doses. The need for multiple doses produces problems with medication administration during school and after-school hours, and therefore with compliance. Previous long-acting stimulants and preparations have shown effects equivalent to twice-daily dosing of MPH. This study tests the efficacy and duration of action, in natural and laboratory settings, of an extended-release MPH preparation designed to last 12 hours and therefore be equivalent to 3-times-daily dosing. METHODS Sixty-eight children with ADHD, 6 to 12 years old, participated in a within-subject, double-blind comparison of placebo, immediate-release (IR) MPH 3 times a day (tid), and Concerta, a once-daily MPH formulation. Three dosing levels of medication were used: 5 mg IR MPH tid/18 mg Concerta once a day (qd); 10 mg IR MPH tid/36 mg Concerta qd; and 15 mg IR MPH tid/54 mg Concerta qd. All children were currently medicated with MPH at enrollment, and each child's dose level was based on that child's MPH dosing before the study. The doses of Concerta were selected to be comparable to the daily doses of MPH that each child received. To achieve the ascending rate of MPH delivery determined by initial investigations to provide the necessary continuous coverage, Concerta doses were 20% higher on a daily basis than a comparable tid regimen of IR MPH. Children received each medication condition for 7 days. The investigation was conducted in the context of a background clinical behavioral intervention in both the natural environment and the laboratory setting. Parents received behavioral parent training and teachers were taught to establish a school-home daily report card (DRC). A DRC is a list of individual target behaviors that represent a child's most salient areas of impairment. Teachers set daily goals for each child's impairment targets, and parents provided rewards at home for goal attainment. Each weekday, teachers completed the DRC, and it was used as a dependent measure of individualized medication response. Teachers and parents also completed weekly standardized ratings of behavior and treatment effectiveness. To evaluate the time course of medication effects, children spent 12 hours in a laboratory setting on Saturdays and medication effects were measured using procedures and methods adapted from our summer treatment program. Measures of classroom behavior and academic productivity/accuracy were taken in a laboratory classroom setting during which children completed independent math and reading worksheets. Measures of social behavior were taken in structured, small-group board game settings and unstructured recess settings. Measures included behavior frequency counts, academic problems completed and accuracy, independent observations, teacher and counselor ratings, and individualized behavioral target goals. Reports of adverse events, sleep quality, and appetite were collected. RESULTS On virtually all measures in all settings, both drug conditions were significantly different from placebo, and the 2 drugs were not different from each other. In children's regular school settings, both medications improved behavior as measured by teacher ratings and individualized target behaviors (the DRC); these effects were seen into the evening as measured by parent ratings. In the laboratory setting, effects of Concerta were equivalent to tid MPH and lasted at least through 12 hours after dosing. Concerta was significantly superior to tid MPH on 2 parent rating scores, and when asked, more parents preferred Concerta than preferred tid IR MPH or placebo. Side effects on children's sleep and appetite were similar for the 2 preparations. In the lab setting, both medications improved productivity and accuracy on arithmetic seatwork assignments, disruptive and on-task behavior, and classroom rule following. Both medications improved children's rule following and negative behavior in small group board games, as well as in unstructured recess settings. Individual target behaviors also showed significant improvement with medication across domains in the laboratory setting. Children's behavior across settings deteriorated across the laboratory day, and the primary effect of medication was to prevent this deterioration as the day wore on. Results support the use of background behavioral treatment in clinical trials of stimulant medication, and illustrate the utility of a measure of individualized daily target goals (ie, the DRC) as an objective measure of medication response in both the laboratory and natural school settings. CONCLUSION This investigation clearly supports the efficacy of the Concerta long-acting formulation of MPH for parents who desire to have medication benefits for their child throughout the day and early evening. (ABSTRACT TRUNCATED)

Title: Cannabinoids in Clinical Practice Content: Cannabis has a potential for clinical use often obscured by unreliable and purely anecdotal reports. The most important natural cannabinoid is the psychoactive tetrahydrocannabinol (Δ9-THC); others include cannabidiol (CBD) and cannabigerol (CBG). Not all the observed effects can be ascribed to THC, and the other constituents may also modulate its action; for example CBD reduces anxiety induced by THC. A standardised extract of the herb may be therefore be more beneficial in practice and clinical trial protocols have been drawn up to assess this. The mechanism of action is still not fully understood, although cannabinoid receptors have been cloned and natural ligands identified. Cannabis is frequently used by patients with multiple sclerosis (MS) for muscle spasm and pain, and in an experimental model of MS low doses of cannabinoids alleviated tremor. Most of the controlled studies have been carried out with THC rather than cannabis herb and so do not mimic the usual clincal situation. Small clinical studies have confirmed the usefulness of THC as an analgesic; CBD and CBG also have analgesic and antiinflammatory effects, indicating that there is scope for developing drugs which do not have the psychoactive properties ofTHC. Patients taking the synthetic derivative nabilone for neurogenic pain actually preferred cannabis herb and reported that it relieved not only pain but the associated depression and anxiety. Cannabinoids are effective in chemotherapy-induced emesis and nabilone has been licensed for this use for several years. Currently, the synthetic cannabinoid HU211 is undergoing trials as a protective agent after brain trauma. Anecdotal reports of cannabis use include case studies in migraine and Tourette’s syndrome, and as a treatment for asthma and glaucoma. Apart from the smoking aspect, the safety profile of cannabis is fairly good. However, adverse reactions include panic or anxiety attacks, which are worse in the elderly and in women, and less likely in children. Although psychosis has been cited as a consequence of cannabis use, an examination of psychiatric hospital admissions found no evidence of this, however, it may exacerbate existing symptoms. The relatively slow elimination from the body of the cannabinoids has safety implications for cognitive tasks, especially driving and operating machinery; although driving impairment with cannabis is only moderate, there is a significant interaction with alcohol. Natural materials are highly variable and multiple components need to be standardised to ensure reproducible effects. Pure natural and synthetic compounds do not have these disadvantages but may not have the overall therapeutic effect of the herb.

Title: Metabolic effects of isoenergetic nutrient exchange over 24 hours in relation to obesity in women. Content: Twenty-four hour whole body indirect calorimetry has been used to study the effects of feeding, during a sedentary test day, isoenergetic diets which varied in fat (3 or 40 per cent of total energy) and carbohydrate (82 or 45 per cent) content. Three groups of women were studied: lean, obese and 'post-obese' after slimming. Energy expenditure was greater in absolute terms in the obese women. Twenty-four hour energy expenditure was lower by only 3-7 per cent when fasting compared to that when fed to achieve energy balance. There were no large differences in energy expenditure between the two diets or between the groups but the thermogenic effect of the high carbohydrate diet was significantly greater than that of the high fat diet (5.8 vs 3.5 per cent of energy expenditure: P less than 0.01). The post-obese tended to have lower energy expenditure per kg FFM than controls when fasting and when high-fat fed, but this pattern was not shown by the obese. Sleeping energy expenditure was particularly low in the post-obese group when high-fat fed. Dirunal variations in RQ appear to show more marked rise in morning RQ from the nocturnal minimum in the obese and post-obese, which might be evidence for an energy-saving mechanism through greater availability of stored dietary carbohydrate.

Title: Modulation of glucose metabolism in macrophages by products of nitric oxide synthase. Content: Nitric oxide (NO) is a product of L-arginine metabolism that suppresses cellular oxidative metabolism through the inhibition of tricarboxylic acid cycle and electron transport chain enzymes. The impact of NO synthase (NOS) activity on specific pathways of glucose metabolism in freshly harvested and overnight-cultured rat resident peritoneal macrophages, at rest and after stimulation with zymosan, was investigated using radiolabeled glucose. NOS activity was modulated through the L-arginine concentration in culture media and the use of its specific inhibitor, NG-monomethyl-L-arginine, and quantitated using radiolabeled L-arginine. Results demonstrated that NOS activity was associated with increased glucose disappearance, glycolysis, and hexose monophosphate shunt activity and, in line with the known inhibition of oxidative metabolism associated with the production of NO, with a decrease in the flux of glucose and butyrate carbon through the tricarboxylic acid cycle. In addition, the relative increase in glucose utilization that follows zymosan stimulation was enhanced by treatments that suppressed NOS activity. These results demonstrate that the characteristics of glucose metabolism by macrophages are, to a significant extent, determined by products of NOS.

Title: Simvastatin reduces platelet-endocardium adhesion in atrial fibrillation. Content: OBJECTIVES To evaluate the relationship between CD40/CD40L system and increased thrombogenesis in AF, and to test the effects of simvastatin treatment. METHODS In vitro study using human tissue, University Hospital (tertiary referral center). Experiments on right atrial segments obtained before the onset of cardiopulmonary bypass were done in either presence or absence of 5 microM simvastatin. Two groups of patients in either chronic atrial fibrillation or sinus rhythm at the time of cardiac surgery. The endocardial expression of CD40, the release of CD40L, and adhesion of platelets to endocardium. Additionally, the thickness of platelet aggregates and the platelet distribution on the endocardium were also evaluated. RESULTS Atrial fibrillation was associated with a significant increase of endocardial CD40 expression (293.1+/-55.1 pg/ml vs. 230.9+/-53.3 pg/ml, p<0.01), and platelet-endocardial adhesion compared with sinus rhythm atria (10.8+/-2.2 vs. 5.2+/-1.3 platelet CD41 AU p<0.01). At immunofluorescence about 62% of fibrillating endocardium was covered by platelets, compared with 12% of not sinus rhythm atria. Addition of simvastatin significantly reduced CD40 expression as well as platelet adhesion to fibrillating atria; its efficacy was not reversed by the addition of mevalonic acid. CONCLUSIONS Chronic atrial fibrillation acutely upregulates CD40 expression as well as platelet adhesion to the endocardium. Simvastatin is effective in modulating this expression, thus it may potentially contribute to reduce the risk of intra-atrial thrombus formation.

Title: Acute and chronic exercise in patients with heart failure with reduced ejection fraction: evidence of structural and functional plasticity and intact angiogenic signalling in skeletal muscle Content: KEY POINTS The vascular endothelial growth factor (VEGF) responses to acute submaximal exercise and training effects in patients with heart failure with reduced ejection fraction (HFrEF) were investigated. Six patients and six healthy matched controls performed knee-extensor exercise (KE) at 50% of maximum work rate before and after (only patients) KE training. Muscle biopsies were taken to assess skeletal muscle structure and the angiogenic response. Before training, during this submaximal KE exercise, patients with HFrEF exhibited higher leg vascular resistance and greater noradrenaline spillover. Skeletal muscle structure and VEGF response were generally not different between groups. Following training, resistance was no longer elevated and noradrenaline spillover was curtailed in the patients. Although, in the trained state, VEGF did not respond to acute exercise, capillarity was augmented. Muscle fibre cross-sectional area and percentage area of type I fibres increased and mitochondrial volume density exceeded that of controls. Structural/functional plasticity and appropriate angiogenic signalling were observed in skeletal muscle of patients with HFrEF. ABSTRACT This study examined the response to acute submaximal exercise and the effect of training in patients with heart failure with reduced ejection fraction (HFrEF). The acute angiogenic response to submaximal exercise in HFrEF after small muscle mass training is debated. The direct Fick method, with vascular pressures, was performed across the leg during knee-extensor exercise (KE) at 50% of maximum work rate (WRmax ) in patients (n = 6) and controls (n = 6) and then after KE training in patients. Muscle biopsies facilitated the assessment of skeletal muscle structure and vascular endothelial growth factor (VEGF) mRNA levels. Prior to training, HFrEF exhibited significantly higher leg vascular resistance (LVR) (≈15%) and significantly greater noradrenaline spillover (≈385%). Apart from mitochondrial volume density, which was significantly lower (≈22%) in HFrEF, initial skeletal muscle structure, including capillarity, was not different between groups. Resting VEGF mRNA levels, and the increase with exercise, was not different between patients and controls. Following training, LVR was no longer elevated and noradrenaline spillover was curtailed. Skeletal muscle capillarity increased with training, as assessed by capillary-to-fibre ratio (≈13%) and number of capillaries around a fibre (NCAF ) (≈19%). VEGF mRNA was now not significantly increased by acute exercise. Muscle fibre cross-sectional area and percentage area of type I fibres both increased significantly with training (≈18% and ≈21%, respectively), while the percentage area of type II fibres fell significantly (≈11%), and mitochondrial volume density now exceeded that of controls. These data reveal structural and functional plasticity and appropriate angiogenic signalling in skeletal muscle of HFrEF patients.

Title: Endothelin-1 is induced by cytokines in human vascular smooth muscle cells: evidence for intracellular endothelin-converting enzyme. Content: Endothelin-1 (ET-1) is the predominant endothelin isopeptide generated by the vascular wall and therefore appears to be the most important peptide involved in regulation of cardiovascular events. Many pathologic conditions are associated with elevations of ET-1 in the blood vessel wall. Because these conditions are often cytokine driven, we examined the effects of a mixture of cytokines on ET-1 production in human vascular smooth muscle cells (VSMCs) derived from internal mammary artery and saphenous vein (SV). Incubation of IMA and SV VSMCs with tumor necrosis factor-alpha (10 ng/ml) and interferon-gamma (1000 U/ml) in combination for up to 48 h markedly elevated the expression of mRNA for prepro-ET-1 and the release of ET-1 into the culture medium. This cytokine-stimulated release of ET-1 was inhibited by a series of dual endothelin-converting enzyme (ECE)/neutral endopeptidase inhibitors, phosphoramidon, CGS 26303, and CGS 26393, with an accompanying increase in big ET-1 release but with no effect on expression of mRNA for prepro-ET-1. These same compounds were 10 times more potent at inhibiting the conversion of exogenously applied big ET-1 to ET-1. ECE-1b/c mRNA is present in SV VSMCs, however no ECE-1a is present in these cells. Thus VSMCs most probably contain, like endothelial cells, an intracellular ECE responsible for the endogenous synthesis of ET-1. Under the influence of pro-inflammatory mediators the vascular smooth muscle can therefore become an important site of ET-1 production, as has already been established for the dilator mediators nitric oxide, prostaglandin I2, and prostaglandin E2.

Title: Epigenetic therapy activates type I interferon signaling in murine ovarian cancer to reduce immunosuppression and tumor burden. Content: Ovarian cancer is the most lethal of all gynecological cancers, and there is an urgent unmet need to develop new therapies. Epithelial ovarian cancer (EOC) is characterized by an immune suppressive microenvironment, and response of ovarian cancers to immune therapies has thus far been disappointing. We now find, in a mouse model of EOC, that clinically relevant doses of DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi, respectively) reduce the immune suppressive microenvironment through type I IFN signaling and improve response to immune checkpoint therapy. These data indicate that the type I IFN response is required for effective in vivo antitumorigenic actions of the DNMTi 5-azacytidine (AZA). Through type I IFN signaling, AZA increases the numbers of CD45+ immune cells and the percentage of active CD8+ T and natural killer (NK) cells in the tumor microenvironment, while reducing tumor burden and extending survival. AZA also increases viral defense gene expression in both tumor and immune cells, and reduces the percentage of macrophages and myeloid-derived suppressor cells in the tumor microenvironment. The addition of an HDACi to AZA enhances the modulation of the immune microenvironment, specifically increasing T and NK cell activation and reducing macrophages over AZA treatment alone, while further increasing the survival of the mice. Finally, a triple combination of DNMTi/HDACi plus the immune checkpoint inhibitor α-PD-1 provides the best antitumor effect and longest overall survival, and may be an attractive candidate for future clinical trials in ovarian cancer.

Title: Antidepressant fluoxetine enhances glucocorticoid receptor function in vitro by modulating membrane steroid transporters. Content: 1. Incubation of LMCAT fibroblast cells with antidepressants potentiates glucocorticoid receptor (GR)-mediated gene transcription in the presence of dexamethasone and cortisol, but not of corticosterone. We have shown that antidepressants do so by inhibiting the LMCAT cell membrane steroid transporter (which is virtually identical to the multidrug resistance P-glycoprotein) and thus by increasing dexamethasone or cortisol intracellular concentrations. However, previous experiments with the antidepressant fluoxetine in the presence of dexamethasone have produced negative results (Pariante et al. (2001). Br. J. Pharmacol., 134, 1335-1343). 2. We have since re-examined the effects of fluoxetine on GR-mediated gene transcription in the presence of dexamethasone. Moreover, we have examined the effects of fluoxetine on GR-mediated gene transcription in the presence of cortisol and corticosterone, and on the intracellular accumulation of radioactive cortisol and corticosterone. Finally, we have examined the effects of fluoxetine on inhibition of P-glycoprotein activity in Caco-2 cells. 3. We now find that fluoxetine (1-10 micro M) enhances GR-mediated gene transcription in the presence of dexamethasone and cortisol (+140-170%), but not of corticosterone, and increases the intracellular accumulation of (3)H-cortisol (+5-15%), but not of (3)H-corticosterone. Moreover, fluoxetine (10 micro M) induces approximately 30% inhibition of PGP activity in Caco-2 cells. 4. Our results show that fluoxetine, like other antidepressants, inhibits membrane steroid transporters.

Title: Intimal smooth muscle cell proliferation after balloon catheter injury. The role of basic fibroblast growth factor. Content: The localization and synthesis of basic fibroblast growth factor (bFGF) in the rat carotid artery were investigated at times of chronic smooth muscle cell proliferation. Immunocytochemical staining showed the presence of bFGF in the uninjured arterial wall, and after balloon injury, this cellular staining was decreased. Western and northern blot analyses likewise showed that the amount of bFGF protein and mRNA decreased after injury. A neutralizing antibody to bFGF was administered 4 and 5 days after injury and was found to have no effect on intimal smooth muscle cell proliferation. These data suggest that an increase in the expression of bFGF is not necessary for chronic smooth muscle cell proliferation observed after balloon catheter injury and that bFGF is not the major mitogen responsible for intimal smooth muscle cell proliferation.

Title: The effects of LY2405319, an FGF21 analog, in obese human subjects with type 2 diabetes. Content: Fibroblast growth factor 21 (FGF21) is a recently discovered metabolic regulator. Exogenous FGF21 produces beneficial metabolic effects in animal models; however, the translation of these observations to humans has not been tested. Here, we studied the effects of LY2405319 (LY), a variant of FGF21, in a randomized, placebo-controlled, double-blind proof-of-concept trial in patients with obesity and type 2 diabetes. Patients received placebo or 3, 10, or 20 mg of LY daily for 28 days. LY treatment produced significant improvements in dyslipidemia, including decreases in low-density lipoprotein cholesterol and triglycerides and increases in high-density lipoprotein cholesterol and a shift to a potentially less atherogenic apolipoprotein concentration profile. Favorable effects on body weight, fasting insulin, and adiponectin were also detected. However, only a trend toward glucose lowering was observed. These results indicate that FGF21 is bioactive in humans and suggest that FGF21-based therapies may be effective for the treatment of selected metabolic disorders.

Title: Effects of Bifidobacterium animalis administered during lactation on allergic and autoimmune responses in rodents. Content: Probiotics are promoted as being beneficial to health and positive effects on the immune system have been reported. Beneficial immune effects have been attributed to several mechanisms, including stimulating T helper 1 (Th1) immunity. To explore the effects of the probiotic Bifidobacterium animalis on Th1- and Th2-mediated immune responses, two different animal models representing either Th1- or Th2-mediated immune responses were used: a rat model for experimental autoimmune encephalomyelitis (EAE) (Th1) and a mouse model for respiratory allergy induced by ovalbumin (OVA) (Th2). B. animalis administration started when the mice or rats were 2 weeks old. Respiratory allergy or EAE were induced when the animals were 6-7 weeks old. In the allergy model, B. animalis modestly reduced the number of infiltrating eosinophils and lymphocytes in the lungs, but no effects on allergen-specific serum immunoglobulin E levels were found. Cytokine profiles assessed after culturing spleen cells with the mitogen concanvalin A (ConA) showed that B. animalis skewed the Th1/Th2 balance towards Th1 in females. However, allergen-induced cytokine production in females was not affected by B. animalis. In males, B. animalis significantly decreased ConA-induced interleukin-13 and a trend towards lower levels of OVA-induced Th2 cytokines. In the EAE model, B. animalis significantly reduced the duration of clinical symptoms by almost 2 days in males and improved the body weight gain during the experimental period compared with the control group. Our data show that B. animalis reduced several immune parameters in the allergy as well as in the autoimmunity model.

Title: Effect of glucosamine on pain-related disability in patients with chronic low back pain and degenerative lumbar osteoarthritis: a randomized controlled trial. Content: CONTEXT Chronic low back pain (LBP) with degenerative lumbar osteoarthritis (OA) is widespread in the adult population. Although glucosamine is increasingly used by patients with chronic LBP, little is known about its effect in this setting. OBJECTIVE To investigate the effect of glucosamine in patients with chronic LBP and degenerative lumbar OA. DESIGN, SETTING, AND PARTICIPANTS A double-blind, randomized, placebo-controlled trial conducted at Oslo University Hospital Outpatient Clinic, Oslo, Norway, with 250 patients older than 25 years of age with chronic LBP (>6 months) and degenerative lumbar OA. INTERVENTIONS Daily intake of 1500 mg of oral glucosamine (n = 125) or placebo (n = 125) for 6 months, with assessment of effect after the 6-month intervention period and at 1 year (6 months postintervention). MAIN OUTCOME MEASURES The primary outcome was pain-related disability measured with the Roland Morris Disability Questionnaire (RMDQ). Secondary outcomes were numerical scores from pain-rating scales of patients at rest and during activity, and the quality-of-life EuroQol-5 Dimensions (EQ-5D) instrument. Data collection occurred during the intervention period at baseline, 6 weeks, 3 and 6 months, and again 6 months following the intervention at 1 year. Group differences were analyzed using linear mixed models analysis. RESULTS At baseline, mean RMDQ scores were 9.2 (95% confidence interval [CI], 8.4-10.0) for glucosamine and 9.7 (95% CI, 8.9-10.5) for the placebo group (P = .37). At 6 months, the mean RMDQ score was the same for the glucosamine and placebo groups (5.0; 95% CI, 4.2-5.8). At 1 year, the mean RMDQ scores were 4.8 (95% CI, 3.9-5.6) for glucosamine and 5.5 (95% CI, 4.7-6.4) for the placebo group. No statistically significant difference in change between groups was found when assessed after the 6-month intervention period and at 1 year: RMDQ (P = .72), LBP at rest (P = .91), LBP during activity (P = .97), and quality-of-life EQ-5D (P = .20). Mild adverse events were reported in 40 patients in the glucosamine group and 46 in the placebo group (P = .48). CONCLUSIONS Among patients with chronic LBP and degenerative lumbar OA, 6-month treatment with oral glucosamine compared with placebo did not result in reduced pain-related disability after the 6-month intervention and after 1-year follow-up. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00404079.

Title: Oxidative damage targets complexes containing DNA methyltransferases, SIRT1, and polycomb members to promoter CpG Islands. Content: Cancer cells simultaneously harbor global losses and gains in DNA methylation. We demonstrate that inducing cellular oxidative stress by hydrogen peroxide treatment recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of the polycomb repressive complex 4. Hydrogen peroxide treatment causes relocalization of these proteins from non-GC-rich to GC-rich areas. Key components are similarly enriched at gene promoters in an in vivo colitis model. Although high-expression genes enriched for members of the complex have histone mark and nascent transcription changes, CpG island-containing low-expression genes gain promoter DNA methylation. Thus, oxidative damage induces formation and relocalization of a silencing complex that may explain cancer-specific aberrant DNA methylation and transcriptional silencing.

Title: Metformin attenuates blood-brain barrier disruption in mice following middle cerebral artery occlusion Content: BACKGROUND Metformin, a widely used hypoglycemic drug, reduces stroke incidence and alleviates chronic inflammation in clinical trials. However, the effect of metformin in ischemic stroke is unclear. Here, we investigated the effect of metformin on ischemic stroke in mice and further explored the possible underlying mechanisms. METHODS Ninety-eight adult male CD-1 mice underwent 90-minute transient middle cerebral artery occlusion (tMCAO). Metformin (200 mg/kg) was administrated for up to 14 days. Neurobehavioral outcomes, brain infarct volume, inflammatory factors, blood-brain barrier (BBB) permeability and AMPK signaling pathways were evaluated following tMCAO. Oxygen glucose deprivation was performed on bEND.3 cells to explore the mechanisms of metformin in inhibiting inflammatory signaling pathways. RESULTS Infarct volume was reduced in metformin-treated mice compared to the control group following tMCAO (P < 0.05). Neurobehavioral outcomes were greatly improved in metformin-treated mice (P < 0.05). MPO+ cells, Gr1+ cells, MPO activity and BBB permeability were decreased after metformin administration (P < 0.05). In addition, metformin activated AMPK phosphorylation, inhibited NF-κB activation, down-regulated cytokine (IL-1β, IL-6, TNF-α) and ICAM-1 expression following tMCAO (P < 0.05). Furthermore, metformin activated AMPK signaling pathway and alleviated oxygen-glucose deprivation-induced ICAM-1 expression in bEND.3 cells (P < 0.05). Compound C, a selective AMPK inhibitor, eliminated this promotional effect. CONCLUSIONS Metformin down-regulated ICAM-1 in an AMPK-dependent manner, which could effectively prevent ischemia-induced brain injury by alleviating neutrophil infiltration, suggesting that metformin is a promising therapeutic agent in stroke therapy.

Title: Effect of Duration and Intermittency of Rifampin on Tuberculosis Treatment Outcomes: A Systematic Review and Meta-Analysis Content: BACKGROUND Treatment regimens for active tuberculosis (TB) that are intermittent, or use rifampin during only the initial phase, offer practical advantages, but their efficacy has been questioned. We conducted a systematic review of treatment regimens for active TB, to assess the effect of duration and intermittency of rifampin use on TB treatment outcomes. METHODS AND FINDINGS PubMed, Embase, and the Cochrane CENTRAL database for clinical trials were searched for randomized controlled trials, published in English, French, or Spanish, between 1965 and June 2008. Selected studies utilized standardized treatment with rifampin-containing regimens. Studies reported bacteriologically confirmed failure and/or relapse in previously untreated patients with bacteriologically confirmed pulmonary TB. Pooled cumulative incidences of treatment outcomes and association with risk factors were computed with stratified random effects meta-analyses. Meta-regression was performed using a negative binomial regression model. A total of 57 trials with 312 arms and 21,472 participants were included in the analysis. Regimens utilizing rifampin only for the first 1-2 mo had significantly higher rates of failure, relapse, and acquired drug resistance, as compared to regimens that used rifampin for 6 mo. This was particularly evident when there was initial drug resistance to isoniazid, streptomycin, or both. On the other hand, there was little evidence of difference in failure or relapse with daily or intermittent schedules of treatment administration, although there was insufficient published evidence of the efficacy of twice-weekly rifampin administration throughout therapy. CONCLUSIONS TB treatment outcomes were significantly worse with shorter duration of rifampin, or with initial drug resistance to isoniazid and/or streptomycin. Treatment outcomes were similar with all intermittent schedules evaluated, but there is insufficient evidence to support administration of treatment twice weekly throughout therapy.

Title: 6-Sulphated Chondroitins Have a Positive Influence on Axonal Regeneration Content: Chondroitin sulphate proteoglycans (CSPGs) upregulated in the glial scar inhibit axon regeneration via their sulphated glycosaminoglycans (GAGs). Chondroitin 6-sulphotransferase-1 (C6ST-1) is upregulated after injury leading to an increase in 6-sulphated GAG. In this study, we ask if this increase in 6-sulphated GAG is responsible for the increased inhibition within the glial scar, or whether it represents a partial reversion to the permissive embryonic state dominated by 6-sulphated glycosaminoglycans (GAGs). Using C6ST-1 knockout mice (KO), we studied post-injury changes in chondroitin sulphotransferase (CSST) expression and the effect of chondroitin 6-sulphates on both central and peripheral axon regeneration. After CNS injury, wild-type animals (WT) showed an increase in mRNA for C6ST-1, C6ST-2 and C4ST-1, but KO did not upregulate any CSSTs. After PNS injury, while WT upregulated C6ST-1, KO showed an upregulation of C6ST-2. We examined regeneration of nigrostriatal axons, which demonstrate mild spontaneous axon regeneration in the WT. KO showed many fewer regenerating axons and more axonal retraction than WT. However, in the PNS, repair of the median and ulnar nerves led to similar and normal levels of axon regeneration in both WT and KO. Functional tests on plasticity after the repair also showed no evidence of enhanced plasticity in the KO. Our results suggest that the upregulation of 6-sulphated GAG after injury makes the extracellular matrix more permissive for axon regeneration, and that the balance of different CSs in the microenvironment around the lesion site is an important factor in determining the outcome of nervous system injury.

Title: Renal outcomes with different fixed-dose combination therapies in patients with hypertension at high risk for cardiovascular events (ACCOMPLISH): a prespecified secondary analysis of a randomised controlled trial. Content: BACKGROUND The Avoiding Cardiovascular Events through Combination Therapy in Patients Living with Systolic Hypertension (ACCOMPLISH) trial showed that initial antihypertensive therapy with benazepril plus amlodipine was superior to benazepril plus hydrochlorothiazide in reducing cardiovascular morbidity and mortality. We assessed the effects of these drug combinations on progression of chronic kidney disease. METHODS ACCOMPLISH was a double-blind, randomised trial undertaken in five countries (USA, Sweden, Norway, Denmark, and Finland). 11 506 patients with hypertension who were at high risk for cardiovascular events were randomly assigned via a central, telephone-based interactive voice response system in a 1:1 ratio to receive benazepril (20 mg) plus amlodipine (5 mg; n=5744) or benazepril (20 mg) plus hydrochlorothiazide (12.5 mg; n=5762), orally once daily. Drug doses were force-titrated for patients to attain recommended blood pressure goals. Progression of chronic kidney disease, a prespecified endpoint, was defined as doubling of serum creatinine concentration or end-stage renal disease (estimated glomerular filtration rate <15 mL/min/1.73 m(2) or need for dialysis). Analysis was by intention to treat (ITT). This trial is registered with ClinicalTrials.gov, number NCT00170950. FINDINGS The trial was terminated early (mean follow-up 2.9 years [SD 0.4]) because of superior efficacy of benazepril plus amlodipine compared with benazepril plus hydrochlorothiazide. At trial completion, vital status was not known for 143 (1%) patients who were lost to follow-up (benazepril plus amlodipine, n=70; benazepril plus hydrochlorothiazide, n=73). All randomised patients were included in the ITT analysis. There were 113 (2.0%) events of chronic kidney disease progression in the benazepril plus amlodipine group compared with 215 (3.7%) in the benazepril plus hydrochlorothiazide group (HR 0.52, 0.41-0.65, p<0.0001). The most frequent adverse event in patients with chronic kidney disease was peripheral oedema (benazepril plus amlodipine, 189 of 561, 33.7%; benazepril plus hydrochlorothiazide, 85 of 532, 16.0%). In patients with chronic kidney disease, angio-oedema was more frequent in the benazepril plus amlodipine group than in the benazepril plus hydrochlorothiazide group. In patients without chronic kidney disease, dizziness, hypokalaemia, and hypotension were more frequent in the benazepril plus hydrochlorothiazide group than in the benazepril plus amlodipine group. INTERPRETATION Initial antihypertensive treatment with benazepril plus amlodipine should be considered in preference to benazepril plus hydrochlorothiazide since it slows progression of nephropathy to a greater extent. FUNDING Novartis.

Title: Effect of hydroxyethyl starch on bleeding after cardiopulmonary bypass: a meta-analysis of randomized trials. Content: OBJECTIVE The effects of hydroxyethyl starch on bleeding after cardiopulmonary bypass were determined. METHODS A meta-analysis was performed of postoperative blood loss in randomized clinical trials of hydroxyethyl starch versus albumin for fluid management in adult cardiopulmonary bypass surgery. Impacts of hydroxyethyl starch molecular weight and molar substitution were assessed. Randomized trials directly comparing different hydroxyethyl starch solutions were also included. RESULTS Eighteen trials with 970 total patients were included. Compared with albumin, hydroxyethyl starch increased postoperative blood loss by 33.3% of a pooled SD (95% confidence interval, 18.2%-48.3%; P < .001). Risk of reoperation for bleeding was more than doubled by hydroxyethyl starch (relative risk, 2.24; 95% confidence interval, 1.14-4.40; P = .020). Hydroxyethyl starch increased transfusion of red blood cells by 28.4% of a pooled SD (95% confidence interval, 12.2%-44.6%; P < .001), of fresh-frozen plasma by 30.6% (95% confidence interval, 8.0%-53.1%; P = .008), and of platelets by 29.8% (95% confidence interval, 3.4%-56.2%; P = .027). None of these effects differed significantly between hydroxyethyl starch 450/0.7 and 200/0.5. Insufficient data were available for hydroxyethyl starch 130/0.4 versus albumin; however, no significant differences were detected in head-to-head comparisons of hydroxyethyl starch 130/0.4 with 200/0.5. Albumin improved hemodynamics. There were no differences in fluid balance, ventilator time, intensive care unit stay, or mortality. CONCLUSIONS Hydroxyethyl starch increased blood loss, reoperation for bleeding, and blood product transfusion after cardiopulmonary bypass. There was no evidence that these risks could be mitigated by lower molecular weight and substitution.

Title: Metformin Normalizes Type 2 Diabetes-Induced Decrease in Cell Proliferation and Neuroblast Differentiation in the Rat Dentate Gyrus Content: In this study, we observed the effects of metformin, one of the most widely prescribed drugs for the treatment of type 2 diabetes, on cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus (SZDG) in Zucker diabetic fatty (ZDF) rats, which are a model for type 2 diabetes. For this, metformin was administered orally once a day to 14-week-old ZDF rats for 2 weeks and the animals were sacrificed at 16 weeks of age. During this period, blood glucose levels were higher in the vehicle-treated ZDF rats than in the Zucker lean control (ZLC) rats. Metformin treatment significantly decreased the blood glucose levels from 15.5 weeks of age. In the SZDG, Ki67 (a marker for cell proliferation)- and doublecortin (DCX, a marker for differentiated neuroblasts)-immunoreactive cells were much lower in the vehicle-treated ZDF rats than in the ZLC rats. In the metformin-treated ZDF group, Ki67- and DCX-immunoreactive cells were significantly increased in the SZDG compared to those in the vehicle-treated ZDF group. These results suggest that diabetes significantly reduces cell proliferation and neuroblast differentiation in the SZDG and that metformin treatment normalizes the reduction of cell proliferation and neuroblast differentiation in the SZDG in diabetic rats.

Title: Effects of Abciximab on the architecture of platelet-rich clots in patients with acute myocardial infarction undergoing primary coronary intervention. Content: BACKGROUND Abciximab plus aspirin improves the TIMI 3 flow rate of the infarct-related artery in patients treated with either percutaneous coronary intervention or thrombolysis. The present study investigated whether the reperfusion efficacy of abciximab relates to modifications of clot architecture in patients admitted for acute myocardial infarction (AMI). METHODS AND RESULTS A total of 23 AMI patients in the Abciximab before Direct angioplasty and stenting in Myocardial Infarction Regarding Acute and Long term follow-up (ADMIRAL) trial received, in a double-blind fashion, either abciximab (n=13) or placebo (n=10) before primary stenting. Viscoelastic (G' in dyne/cm(2)) and morphological (mean platelet aggregate surface area [SAG] in micrometer(2)) indexes of ex vivo platelet-rich clots (PRC) were assessed in a double-blind fashion before and after the bolus administration of abciximab or placebo. G' and SAG reflect the mechanical and morphological impact of activated platelets on the PRC fibrin network, respectively. Abciximab administration reduced G' by 63% (P=0.0001) and SAG by 65% (P=0.0007), and no effect was seen in the placebo group. These abciximab-related changes increased fibrin exposure as a consequence of the platelet-aggregate surface reduction and may have improved endogenous fibrinolysis. These effects were identified in all patients, independent of previous heparin administration. CONCLUSIONS Abciximab dramatically reduces platelet aggregate size and increases the fibrin accessibility of ex vivo PRC in AMI patients. These modifications could participate in the better coronary artery patency observed with abciximab.

Title: Protein S-nitrosylation: a physiological signal for neuronal nitric oxide Content: Nitric oxide (NO) has been linked to numerous physiological and pathophysiological events that are not readily explained by the well established effects of NO on soluble guanylyl cyclase. Exogenous NO S-nitrosylates cysteine residues in proteins, but whether this is an important function of endogenous NO is unclear. Here, using a new proteomic approach, we identify a population of proteins that are endogenously S-nitrosylated, and demonstrate the loss of this modification in mice harbouring a genomic deletion of neuronal NO synthase (nNOS). Targets of NO include metabolic, structural and signalling proteins that may be effectors for neuronally generated NO. These findings establish protein S-nitrosylation as a physiological signalling mechanism for nNOS.

Title: Differential Effects of N-Acetylcysteine, Theophylline or Bicarbonate on Contrast-Induced Rat Renal Vasoconstriction Content: Background: Vasoconstriction and reactive oxygen species (ROS) accumulation following contrast media (CM) injection are the key factors triggering CM-induced nephropathy. We compared the effects of N-acetylcysteine (NAC), theophylline or sodium bicarbonate on intrarenal vasoconstriction and ROS generation in a rat model of CM-induced nephropathy. Methods: Following a 3-day dehydration, Sprague-Dawley rats received CM (Telebrix) or sham ‘CM’ injection of 0.9% saline. Part of them received NAC, theophylline or bicarbonate prior to CM. Medullar renal blood flow was estimated by laser Doppler. The animals were sacrificed 1, 15 or 30 min after the respective treatments, their kidneys allocated and intrarenal STAT-8 isoprostane, PGE2 and NO assessed. Results: Vasoconstriction was significantly attenuated by NAC. Theophylline only mildly attenuated the perfusion drop at 15 min, and was ineffective following 30 min. Unlike theophylline or bicarbonate, NAC significantly augmented intrarenal PGE2. NAC, theophylline but not bicarbonate, gradually increased intrarenal NO. In all experimental variables, CM-induced ROS accumulation, represented by STAT-8 isoprostane estimation, progressed undisturbed. Conclusions: (1) CM-induced intrarenal vasoconstriction was efficiently prohibited by NAC but not bicarbonate or theophylline; (2) the vasodilatory effect of NAC was mediated via increased PGE2 synthesis, and (3) ROS accumulation was a primary renal response to CM-induced injury, not affected by any pharmacologic manipulations.

Title: The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Content: Recent studies suggest that statins can function to protect the vasculature in a manner that is independent of their lipid-lowering activity. We show here that statins rapidly activate the protein kinase Akt/PKB in endothelial cells. Accordingly, simvastatin enhanced phosphorylation of the endogenous Akt substrate endothelial nitric oxide synthase (eNOS), inhibited apoptosis and accelerated vascular structure formation in vitro in an Akt-dependent manner. Similar to vascular endothelial growth factor (VEGF) treatment, both simvastatin administration and enhanced Akt signaling in the endothelium promoted angiogenesis in ischemic limbs of normocholesterolemic rabbits. Therefore, activation of Akt represents a mechanism that can account for some of the beneficial side effects of statins, including the promotion of new blood vessel growth.

Title: Wnt7a/Fzd7 Signalling Directly Activates the Akt/mTOR Anabolic Growth Pathway in Skeletal Muscle Content: Wnt7a signals through its receptor Fzd7 to activate the planar-cell-polarity pathway and drive the symmetric expansion of satellite stem cells resulting in enhanced repair of skeletal muscle. In differentiated myofibres, we observed that Wnt7a binding to Fzd7 directly activates the Akt/mTOR growth pathway, thereby inducing myofibre hypertrophy. Notably, the Fzd7 receptor complex was associated with Gα(s) and PI(3)K and these components were required for Wnt7a to activate the Akt/mTOR growth pathway in myotubes. Wnt7a-Fzd7 activation of this pathway was completely independent of IGF-receptor activation. Together, these experiments demonstrate that Wnt7a-Fzd7 activates distinct pathways at different developmental stages during myogenic lineage progression, and identify a non-canonical anabolic signalling pathway for Wnt7a and its receptor Fzd7 in skeletal muscle.

Title: Bile acid receptor TGR5 agonism induces NO production and reduces monocyte adhesion in vascular endothelial cells. Content: OBJECTIVE TGR5 is a G-protein-coupled receptor for bile acids. So far, little is known about the function of TGR5 in vascular endothelial cells. APPROACH AND RESULTS In bovine aortic endothelial cells, treatment with a bile acid having a high affinity to TGR5, taurolithocholic acid (TLCA), significantly increased NO production. This effect was abolished by small interfering RNA-mediated depletion of TGR5. TLCA-induced NO production was also observed in human umbilical vein endothelial cells measured via intracellular cGMP accumulation. TLCA increased endothelial NO synthase(ser1177) phosphorylation in human umbilical vein endothelial cells. This response was accompanied by increased Akt(ser473) phosphorylation and intracellular Ca(2+). Inhibition of these signals significantly decreased TLCA-induced NO production. We next examined whether TGR5-mediated NO production affects inflammatory responses of endothelial cells. In human umbilical vein endothelial cells, TLCA significantly reduced tumor necrosis factor-α-induced adhesion of monocytes, vascular cell adhesion molecule-1 expression, and activation of nuclear factor-κB. TLCA also inhibited lipopolysaccharide-induced monocyte adhesion to mesenteric venules in vivo. These inhibitory effects of TLCA were abrogated by NO synthase inhibition. CONCLUSIONS TGR5 agonism induces NO production via Akt activation and intracellular Ca(2+) increase in vascular endothelial cells, and this function inhibits monocyte adhesion in response to inflammatory stimuli.

Title: Effect of enalapril on 12-year survival and life expectancy in patients with left ventricular systolic dysfunction: a follow-up study. Content: BACKGROUND In the studies of left ventricular dysfunction (SOLVD), enalapril reduced mortality in patients with symptomatic but not asymptomatic left ventricular systolic dysfunction during the trial. We did a 12-year follow-up of SOLVD to establish if the mortality reduction with enalapril among patients with heart failure was sustained, and whether a subsequent reduction in mortality would emerge among those with asymptomatic ventricular dysfunction. METHODS Of the 6797 patients previously enrolled in the SOLVD prevention and treatment trials, we ascertained the subsequent vital status of 5165 individuals who were alive when the trials had been completed. Follow-up was done through direct contacts in Belgium and linkages with national death registries and federal beneficiary or historic tax summary files in the USA and Canada. FINDINGS Follow-up was 99.8% (6784/6797) complete. In the prevention trial, 50.9% (1074/2111) of the enalapril group had died compared with 56.4% (1195/2117) of the placebo group (generalised Wilcoxon p=0.001). In the treatment trial, 79.8% (1025/1285) of the enalapril group had died compared with 80.8% (1038/1284) of the placebo group (generalised Wilcoxon p=0.01). The reductions in cardiac deaths were significant and similar in both trials. When data for the prevention and treatment trials were combined, the hazard ratio for death was 0.90 for the enalapril group compared with the placebo group (95% CI 0.84-0.95, generalised Wilcoxon p=0.0003). Enalapril extended median survival by 9.4 months in the combined trials (95% CI 2.8-16.5, p=0.004). INTERPRETATION Treatment with enalapril for 3-4 years led to a sustained improvement in survival beyond the original trial period in patients with left ventricular systolic dysfunction, with an important increase in life expectancy.

Title: Enhancing circadian clock function in cancer cells inhibits tumor growth Content: BACKGROUND Circadian clocks control cell cycle factors, and circadian disruption promotes cancer. To address whether enhancing circadian rhythmicity in tumor cells affects cell cycle progression and reduces proliferation, we compared growth and cell cycle events of B16 melanoma cells and tumors with either a functional or dysfunctional clock. RESULTS We found that clock genes were suppressed in B16 cells and tumors, but treatments inducing circadian rhythmicity, such as dexamethasone, forskolin and heat shock, triggered rhythmic clock and cell cycle gene expression, which resulted in fewer cells in S phase and more in G1 phase. Accordingly, B16 proliferation in vitro and tumor growth in vivo was slowed down. Similar effects were observed in human colon carcinoma HCT-116 cells. Notably, the effects of dexamethasone were not due to an increase in apoptosis nor to an enhancement of immune cell recruitment to the tumor. Knocking down the essential clock gene Bmal1 in B16 tumors prevented the effects of dexamethasone on tumor growth and cell cycle events. CONCLUSIONS Here we demonstrated that the effects of dexamethasone on cell cycle and tumor growth are mediated by the tumor-intrinsic circadian clock. Thus, our work reveals that enhancing circadian clock function might represent a novel strategy to control cancer progression.

Title: Topiramate for smoking cessation: a randomized, placebo-controlled pilot study. Content: INTRODUCTION Topiramate (TOP) blocks glutamate receptors and facilitates GABA (γ-aminobutyric acid) neurotransmission, effects that may facilitate smoking cessation. We compared the effects of behavioral counseling combined with (a) TOP, (b) TOP/nicotine patch (TOP/NIC), or (c) placebo (PLC) for smoking cessation. METHODS We conducted a 10-week randomized trial in which subjects and research personnel were blinded to TOP versus PLC but not to the TOP/NIC patch condition. In groups receiving TOP, the medication dosage was titrated gradually up to 200 mg/day. The smoking quit date (QD) was scheduled after 2 weeks of medication treatment. NIC (21 mg) was started on the QD in subjects randomized to the TOP/NIC condition. The main outcome measure was the end-of-treatment, 4-week continuous abstinence rate (CAR; biochemically confirmed). RESULTS Fifty-seven subjects were randomized to treatment. The 4-week CAR was 1 of 19 (5%) in the PLC group, 5 of 19 (26%) in the TOP group, and 7 of 19 (37%) in the TOP/NIC group (p = .056). Pairwise comparisons showed a difference between TOP/NIC and PLC (p = .042) and a nonsignificant difference between TOP and PLC (p = .18). The PLC group gained 0.37 lb/week, the TOP group lost 0.41 lb/week, and the TOP/NIC group lost 0.07 lb/week (p = .004). Pairwise comparisons showed a difference between TOP and PLC (p < .001) and between TOP/NIC and PLC groups (p = .035). Paresthesia was more frequent in subjects on TOP than PLC (p = .011). CONCLUSIONS TOP, alone or in combination with the NIC, resulted in a numerically higher quit rate than PLC and decreased weight. A larger, PLC-controlled trial is needed to confirm these findings.

Title: The atypical antipsychotic olanzapine causes weight gain by targeting serotonin receptor 2C. Content: Atypical antipsychotics such as olanzapine often induce excessive weight gain and type 2 diabetes. However, the mechanisms underlying these drug-induced metabolic perturbations remain poorly understood. Here, we used an experimental model that reproduces olanzapine-induced hyperphagia and obesity in female C57BL/6 mice. We found that olanzapine treatment acutely increased food intake, impaired glucose tolerance, and altered physical activity and energy expenditure in mice. Furthermore, olanzapine-induced hyperphagia and weight gain were blunted in mice lacking the serotonin 2C receptor (HTR2C). Finally, we showed that treatment with the HTR2C-specific agonist lorcaserin suppressed olanzapine-induced hyperphagia and weight gain. Lorcaserin treatment also improved glucose tolerance in olanzapine-fed mice. Collectively, our studies suggest that olanzapine exerts some of its untoward metabolic effects via antagonism of HTR2C.