Structural and functional characterization of G protein-coupled adenosine receptors and the orphan receptor GPR143

The present thesis deals with the characterization of G protein-coupled receptors (GPCRs) and their interaction with modulators on a molecular level. We focused on the structural and functional investigation of different types of GPCRs: the well known, previously crystallized adenosine A2A receptor and its closely related, but much less investigated relative, the adenosine A2B receptor, both of which are promising drug targets, and the underexplored orphan receptor GPR143, an intracellular GPCR which is involved in Ocular Albinism type I. Our goal was to study the impact of structural changes of GPCRs on ligand binding, signaling, and ligand selectivity as well as their allosteric modulation by interaction with other proteins. The results of our studies do not only expand the knowledge of basic biological processes, but they will also contribute to design of potent and selective receptor ligands which have potential as novel therapeutics

Topical use of tranexamic acid in coronary artery bypass operations: A double-blind, prospective, randomized, placebo-controlled study

AbstractObjectives: We sought to investigate the effect of topical application of tranexamic acid into the pericardial cavity in reducing postoperative blood loss in coronary artery surgery. Methods: A prospective, randomized, double-blind investigation with parallel groups was performed. Forty consecutive patients undergoing primary coronary surgery were randomly assigned to group 1 (tranexamic acid group) or group 2 (placebo group). Tranexamic acid (1 g in 100 mL of saline solution) or placebo was poured into the pericardial cavity and over the mediastinal tissues before sternal closure. The drainage of mediastinal blood was measured hourly. Results: Chest tube drainage in the first 24 hours was 485 ± 166 mL in the tranexamic acid group and 641 ± 184 mL in the placebo group (P = .01). Total postoperative blood loss was 573 ± 164 mL and 739 ± 228 mL, respectively (P = .01). The use of banked donor blood products was not significantly different between the two groups. Tranexamic acid could not be detected in any of the blood samples blindly collected from 24 patients to verify whether any systemic absorption of the drug occurred. There were no deaths in either group. None of the patients required reoperation for bleeding. Conclusions: Topical application of tranexamic acid into the pericardial cavity after cardiopulmonary bypass in patients undergoing primary coronary bypass operations significantly reduces postoperative bleeding. Further studies must be carried out to clarify whether a more pronounced effect on both bleeding and blood products requirement might be seen in procedures with a higher risk of bleeding. (J Thorac Cardiovasc Surg 2000;119:575-80

shear modulus of masonry walls a critical review

Abstract In the assessment of seismic performance of masonry buildings, the proper definition of mechanical parameters of masonry, the shear modulus in particular, is a critical issue. Moreover, considering that existing buildings are characterized by several masonry types, depending on the material as well as on the texture, mechanical parameters can vary in a very wide range, also because they depend on many other parameters and in particular on the integrity of the walls and on the stress level. Although the in situ or laboratory experimental evaluation of the G modulus has been the subject of a wide literature concerning flat jacks, diagonal and single compression and shear-compression test results, its outcomes are often contradictory. In effect, values given by different studies often differ significantly, even for the same class of masonry. Since the intrinsic scattering of the parameter is not sufficient by itself to justify the huge variability of the results, a critical discussion of the results as well as of the individual test arrangements is necessary to make the background more reliable, also in view of better addressing further studies- A huge database has been setup combining masonry test results available in the relevant scientific literature with the test results obtained in the framework of the in situ experimental campaign carried out by the authors for the assessment of seismic vulnerability of masonry school buildings in the Municipality of Florence. The analysis of the database underlines that values of the shear modulus G, which is a fundamental parameter for the definition of capacity curve for walls commonly used in non-linear static analysis, are extremely scattered. Testing methodology and arrangement are discussed and a possible procedure is proposed to arrive to sounder estimations of relevant mechanical parameter of existing building masonry

Fine Structure of Glycosaminoglycans from Fresh and Decellularized Porcine Cardiac Valves and Pericardium

Cardiac valves are dynamic structures, exhibiting a highly specialized architecture consisting of cells and extracellular matrix with a relevant proteoglycan and glycosaminoglycan content, collagen and elastic fibers. Biological valve substitutes are obtained from xenogenic cardiac and pericardial tissues. To overcome the limits of such non viable substitutes, tissue engineering approaches emerged to create cell repopulated decellularized scaffolds. This study was performed to determine the glycosaminoglycans content, distribution, and disaccharides composition in porcine aortic and pulmonary valves and in pericardium before and after a detergent-based decellularization procedure. The fine structural characteristics of galactosaminoglycans chondroitin sulfate and dermatan sulfate were examined by FACE. Furthermore, the mechanical properties of decellularized pericardium and its propensity to be repopulated by in vitro seeded fibroblasts were investigated. Results show that galactosaminoglycans and hyaluronan are differently distributed between pericardium and valves and within heart valves themselves before and after decellularization. The distribution of glycosaminoglycans is also dependent from the vascular district and topographic localization. The decellularization protocol adopted resulted in a relevant but not selective depletion of galactosaminoglycans. As a whole, data suggest that both decellularized porcine heart valves and bovine pericardium represent promising materials bearing the potential for future development of tissue engineered heart valve scaffolds

DNA metabarcoding of forensic mycological samples

DNA metabarcoding and massive parallel sequencing are valuable molecular tools for the characterization of environmental samples. In forensic sciences, the analysis of the sample’s fungal population can be highly informative for the estimation of post-mortem interval, the ascertainment of deposition time, the identification of the cause of death, or the location of buried corpses. Unfortunately, metabarcoding data analysis often requires strong bioinformatic capabilities that are not widely available in forensic laboratories. The present paper describes the adoption of a user-friendly cloud-based application for the identification of fungi in typical forensic samples. The samples have also been analyzed through the QIIME pipeline, obtaining a relevant data concordance on top genus classification results (88%).The availability of a user-friendly application that can be run without command line activities will increase the popularity of metabarcoding fungal analysis in forensic samples

Plasma Levels of t-PA and PAI-1 Correlate With the Formation of Experimental Post-Surgical Peritoneal Adhesions

This study has evaluated whether systemic changes of plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) parallel the adhesions development and whether they could be used as predictors of adhesion risk. This has been studied in an animal model of post-surgical peritoneal adhesion by monitoring for 10 days the plasma and tissue levels of t-PA and PAI-1. The results showed that both tissular and plasmatic levels of t-PA were decreased in concomitance with the development of peritoneal adhesions. In contrast, PAI-1 was found increased into the tissue and into the plasma samples of the rats taken at 5 and 10 days time points. Inflammatory mediators such as ICAM-1, VCAM-1, and IL-6 within the peritoneal lavage fluid also correlated with the adhesion formation process. In conclusion, post-surgical peritoneal adhesions provide alterations of local inflammatory components and local and systemic fibrinolytic components, possibly with PAI-1 quenching t-PA. This may have potential for the identification of high-risk patients

Adenosine A2A receptor ligand recognition and signaling is blocked by A2B receptors

The adenosine receptor (AR) subtypes A2A and A2B are rhodopsin-like Gs protein-coupled receptors whose expression is highly regulated under pathological, e.g. hypoxic, ischemic and inflammatory conditions. Both receptors play important roles in inflammatory and neurodegenerative diseases, are blocked by caffeine, and have now become major drug targets in immuno-oncology. By Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC) and proximity ligation assays (PLA) we demonstrated A2A-A2BAR heteromeric complex formation. Moreover we observed a dramatically altered pharmacology of the A2AAR when co-expressed with the A2BAR (A2B ≥ A2A) in recombinant as well as in native cells. In the presence of A2BARs, A2A-selective ligands lost high affinity binding to A2AARs and displayed strongly reduced potency in cAMP accumulation and dynamic mass redistribution (DMR) assays. These results have major implications for the use of A2AAR ligands as drugs as they will fail to modulate the receptor in an A2A-A2B heteromer context. Accordingly, A2A-A2BAR heteromers represent novel pharmacological targets

Metformin Enhances Cisplatin-Induced Apoptosis and Prevents Resistance to Cisplatin in Co-mutated KRAS/LKB1 NSCLC

Abstract Introduction We hypothesized that activating KRAS mutations and inactivation of the liver kinase B1 (LKB1) oncosuppressor can cooperate to sustain NSCLC aggressiveness. We also hypothesized that the growth advantage of KRAS/LKB1 co-mutated tumors could be balanced by higher sensitivity to metabolic stress conditions, such as metformin treatment, thus revealing new strategies to target this aggressive NSCLC subtype. Methods We retrospectively determined the frequency and prognostic value of KRAS/LKB1 co-mutations in tissue specimens from NSCLC patients enrolled in the TAILOR trial. We generated stable LKB1 knockdown and LKB1-overexpressing isogenic H1299 and A549 cell variants, respectively, to test the in vitro efficacy of metformin. We also investigated the effect of metformin on cisplatin-resistant CD133+ cells in NSCLC patient-derived xenografts. Results We found a trend towards worse overall survival in patients with KRAS/LKB1 co-mutated tumors as compared to KRAS-mutated ones (hazard ratio: 2.02, 95% confidence interval: 0.94–4.35, p = 0.072). In preclinical experiments, metformin produced pro-apoptotic effects and enhanced cisplatin anticancer activity specifically in KRAS/LKB1 co-mutated patient-derived xenografts. Moreover, metformin prevented the development of acquired tumor resistance to 5 consecutive cycles of cisplatin treatment (75% response rate with metformin-cisplatin as compared to 0% response rate with cisplatin), while reducing CD133+ cells. Conclusions LKB1 mutations, especially when combined with KRAS mutations, may define a specific and more aggressive NSCLC subtype. Metformin synergizes with cisplatin against KRAS/LKB1 co-mutated tumors, and may prevent or delay the onset of resistance to cisplatin by targeting CD133+ cancer stem cells. This study lays the foundations for combining metformin with standard platinum-based chemotherapy in the treatment of KRAS/LKB1 co-mutated NSCLC