THROMBOXANE PROSTANOID RECEPTOR: FUNCTION, ACTIVATION AND POSSIBLE TARGET FOR CARDIOVASCULAR PROTECTION

Abstract In the last few years cardiovascular diseases are considered one of the major cause of death, and one of the main player is TXA2 (Thromboxane A2), a product of arachidonic acid metabolism generated from the activity of thromboxane synthase on prostaglandin H2 intermediate via cyclooxygenase (COX). TXA2 is responsible for platelets activation and aggregation, thrombus formation, and thus it can cause stroke and myocardial infarction. TXA2 exerts its actions through the TP receptor, a widely expressed GPCR (G protein coupled receptor) present in many cell types among different organ systems. During my thesis I worked to shed light on the mechanism of activation of TP receptor WT (wild type), and two of its mutants (TP\u3b1E129V and TP\u3b1R130V) of the highly conserved motif E/DRY, in order to assign each receptor state to the CTC (Cubic Ternary Complex) model. In particular, using the new technique SPASM (Systematic Protein Affinity Strength Modulation), the goal was to understand the conformational state of TP\u3b1WT and mutants in basal condition, i.e. their coupling or uncoupling states with G proteins. The results obtained suggest that TP\u3b1E129V (SAM-super active mutant) is in an \u2018active-like\u2019 conformation corresponding to the RG state (inactive, coupled to G protein), on the contrary, TP\u3b1R130V (loss of function mutant) seems to display an inactive R conformation (uncoupled to G protein), as envisioned by CTC model. The study of TP\u3b1 receptor induced us to consider a second focus in my thesis: TP\u3b1 receptor as a possible target for new chemical entities with both COX-2 selectivity (COXIB) and TP antagonist activity. New compounds were obtained modulating the structure of existing drugs (lumiracoxib and RC 0) to obtain novel multitarget NSAIDs (Nonsteroidal Anti-inflammatory Drug) endowed with balanced COXIB and TP receptor antagonist properties. Antagonistic activity on the TP receptor was examined for all compounds by evaluating the inhibition of platelets aggregation induced by the stable TXA2 receptor agonist U46619. COX-1 and COX-2 inhibition were assessed in human washed platelets (challenged by the calcium ionophoreA23187) and human lympho-monocytes suspension (stimulated with lipopolysaccharides), respectively. COX selectivity was determined by calculating IC50 values ratio (COX-2/COX-1) obtained from concentration-response curves. Among the lumiracoxib derivatives, the tetrazole compound 18 and the trifluoromethan sulfonamido-isoster 20 were the more active antagonists at the TP receptor, preventing human platelet aggregation and intracellular signalling, with pA2 values statistically higher than lumiracoxib. Comparative data regarding COX- 2/COX-1 selectivity showed that while compounds 18 and 7 were rather potent and selective COX-2 inhibitors, compound 20 was somehow less potent and selective for COX-2. Among the RC 0 derivatives, of particular interest resulted compounds SWE 74, CP 7 and CP 8, because they demonstrated to be fairly selective for COX-2 enzyme, but they appeared to be the weakest TP receptor antagonists among the 35 compounds tested. On the other hand, the last compound of interest was SWE 96, a molecule possessing a good activity on TP\u3b1 receptor, but lacking selectivity in term of COX-2 inhibition, that is behaving like traditional NSAIDs. All the other derivatives tested were not selective COX-2 inhibitors and/or did not inhibit platelet aggregation. Taking advantage of what we learned in terms of structural requirements for COX-2 selective inhibition and TP antagonism, additional studies will certainly be carried out to improve the pharmacodynamic profile of these molecules before a careful evaluation can be considered in an in vivo animal model

Constrained Collaborative Power Management

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Epidemiology of distal radius fractures: a detailed survey on a large sample of patients in a suburban area

Background: Literature lacks data on correlations between epidemiology and clinical data of patients with distal radius fractures (DRFs). Aim: The aim of this study was to present a detailed epidemiologic survey of a large consecutive series of patient with DRFs. Materials and Methods: This retrospective study included 827 consecutive patients (579 females, 248 men) who sustained a DRFs in the last 5 years. All fractures were radiographically evaluated. DRFs were classifed according to Association of Osteosynthesis classifcation. Data on age, gender, side, period in which fracture occurred, and fracture mechanism were collected. Statistical analysis was performed. Results: The patients’ mean age was 60.23 [standard deviation (SD) 16.65] years, with the left side being most frequently involved (56.1%). The mean age of females at the time of fracture was signifcantly higher than that of males. The most frequent pattern of fracture was the complete articular fracture (64.3%), while the most represented fracture type was 2R3A2.2 (21.5%). Regarding the period in which the fracture occurred, 305 DRFs (37.5%) were observed in the warmer months and 272 (33.4%) in the colder months. Low-energy trauma occurring outside home was found to be the major cause of DRF throughout the year. In both genders, trauma mechanism 2 was more frequent (59.4% F; 31.9% M; p<0.01). A bimodal distribution of fracture mechanisms was found in males when considering the patient’s age with a highenergy mechanism of fracture (3 and 4), identifed in 21% (n=52) of males aged 18–45 years, and a low-energy mechanism (1 and 2) was observed in 39.9% (n=99) of males aged>45 years. A signifcant correlation between all trauma mechanisms (from 1 to 6) and diferent fracture patterns (complete, partial, and extraarticular) was found (p value<0.001). The mean age of patients with extraarticular fractures (mean age 61.75 years; SD 18.18 years) was higher than that of those with complete (mean age 59.84 years; SD 15.67 years) and partial fractures (mean age 55.26 years; SD 18.31 years). Furthermore, considering diferent fracture patterns and patient age groups, a statistically signifcant diference was found (p<0.001). Conclusions: DRFs have a higher prevalence in females, an increase in incidence with older age, and no seasonal predisposition. Low-energy trauma occurring at home is the main cause of fracture among younger males sustaining fractures after sports trauma; Complete articular is the most frequent fracture pattern, while 2R3A2.2 is most frequent fracture type. Level of evidence: Level IV; case series; descriptive epidemiology stud

Antidepressant-like effects of pharmacological inhibition of FAAH activity in socially isolated female rats

Pharmacological inhibition of the enzyme fatty acid amide hydrolase (FAAH), which terminates signaling of the endocannabinoid N-arachidonoylethanolamine (or anandamide, AEA), exerts favourable effects in rodent models of stress-related depression. Yet although depression seems to be more common among women than men and in spite of some evidence of sex differences in treatment efficacy, preclinical development of FAAH inhibitors for the pharmacotherapy of stress-related depression has been predominantly conducted in male animals. Here, adult female rats were exposed to six weeks of social isolation and, starting from the second week, treated with the FAAH inhibitor URB694 (0.3 mg/kg/day, i.p.) or vehicle. Compared to pair-housed females, socially isolated female rats treated with vehicle developed behavioral (mild anhedonia, passive stress coping) and physiological (reduced body weight gain, elevated plasma corticosterone levels) alterations. Moreover, prolonged social isolation provoked a reduction in brain-derived neurotrophic factor (BDNF) and AEA levels within the hippocampus. Together, these changes are indicative of an increased risk of developing a depressive-like state. Conversely, pharmacological inhibition of FAAH activity with URB694 restored both AEA and BDNF levels within the hippocampus of socially isolated rats and prevented the development of behavioral and physiological alterations. These results suggest a potential interplay between AEA-mediated signaling and hippocampal BDNF in the pathogenesis of depression-relevant behaviors and physiological alterations and antidepressant action of FAAH inhibition in socially isolated female rats

Surgical management of breast cancer in BRCA mutation carriers: A single centre experience

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Lactobacillus rhamnosus lowers zebrafish lipid content by changing gut microbiota and host transcription of genes involved in lipid metabolism.

The microbiome plays an important role in lipid metabolism but how the introduction of probiotic communities affects host lipid metabolism is poorly understood. Using a multidisciplinary approach we addressed this knowledge gap using the zebrafish model by coupling high-throughput sequencing with biochemical, molecular and morphological analysis to evaluate the changes in the intestine. Analysis of bacterial 16S libraries revealed that Lactobacillus rhamnosus was able to modulate the gut microbiome of zebrafish larvae, elevating the abundance of Firmicutes sequences and reducing the abundance of Actinobacteria. The gut microbiome changes modulated host lipid processing by inducing transcriptional down-regulation of genes involved in cholesterol and triglycerides metabolism (fit2, agpat4, dgat2, mgll, hnf4α, scap, and cck) concomitantly decreasing total body cholesterol and triglyceride content and increasing fatty acid levels. L. rhamnosus treatment also increased microvilli and enterocyte lengths and decreased lipid droplet size in the intestinal epithelium. These changes resulted in elevated zebrafish larval growth. This integrated system investigation demonstrates probiotic modulation of the gut microbiome, highlights a novel gene network involved in lipid metabolism, provides an insight into how the microbiome regulates molecules involved in lipid metabolism, and reveals a new potential role for L. rhamnosus in the treatment of lipid disorders

Comparison of amsler–krumeich and sandali classifications for staging eyes with keratoconus

Keratoconus (KC) is the most common corneal ectasia characterized by progressive corneal thinning, protrusion, and irregular astigmatism. The Amsler–Krumeich classification based on the analysis of corneal topography, corneal thickness, refraction and biomicroscopy is the most commonly used; recently, a new classification based on anterior segment Optical Coherence Tomography was introduced by Sandali and colleagues. Since there is no information about the possible agreement between these two classifications, the aim of this study is to compare the stratification of consecutive KC patients using the Amsler–Krumeich and Sandali classifications, and to further ascertain KC cases in which one classification is preferred over the other. Overall, 252 eyes of 137 patients (41.45 ± 16.93 years) were analyzed: in 156 eyes (61.9%), the Amsler and Sandali staging differed in one stage while in 75 cases (29.8%) it differed in two or more stages. In 222 eyes (88.1%), the Sandali staging was higher compared to the Amsler one. These results show that the two classifications are not fully interchangeable: the Amsler–Krumeich classification is more appropriate in identifying and longitudinally monitoring patients with early stages of KC, while the Sandali classification for the diagnosis and follow-up of patients with more advanced stages, particularly when a surgical planning has to be chosen

Exercise Intensity Modulation of Hepatic Lipid Metabolism

Lipid metabolism in the liver is complex and involves the synthesis and secretion of very low density lipoproteins (VLDL), ketone bodies, and high rates of fatty acid oxidation, synthesis, and esterification. Exercise training induces several changes in lipid metabolism in the liver and affects VLDL secretion and fatty acid oxidation. These alterations are even more conspicuous in disease, as in obesity, and cancer cachexia. Our understanding of the mechanisms leading to metabolic adaptations in the liver as induced by exercise training has advanced considerably in the recent years, but much remains to be addressed. More recently, the adoption of high intensity exercise training has been put forward as a means of modulating hepatic metabolism. The purpose of the present paper is to summarise and discuss the merit of such new knowledge

Comparison of anti-inflammatory and clinical effects of beclomethasone dipropionate and salmeterol in moderate asthma

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