Identification of Novel GSK-3ß Hits Using Competitive Biophysical Assays.

Glycogen synthase kinase 3 beta (GSK-3ß) is an evolutionarily conserved serine-threonine kinase dysregulated in numerous pathologies, such as Alzheimer's disease and cancer. Even though GSK-3ß is a validated pharmacological target most of its inhibitors have two main limitations: the lack of selectivity due to the high homology that characterizes the ATP binding site of most kinases, and the toxicity that emerges from GSK-3ß complete inhibition which translates into the impairment of the plethora of pathways GSK-3ß is involved in. Starting from a 1D 19F NMR fragment screening, we set up several biophysical assays for the identification of GSK-3ß inhibitors capable of binding protein hotspots other than the ATP binding pocket or to the ATP binding pocket, but with an affinity able of competing with a reference binder. A phosphorylation activity assay on a panel of several kinases provided selectivity data that were further rationalized and corroborated by structural information on GSK-3ß in complex with the hit compounds. In this study, we identified promising fragments, inhibitors of GSK-3ß, while proposing an alternative screening workflow that allows facing the flaws that characterize the most common GSK-3ß inhibitors through the identification of selective inhibitors and/or inhibitors able to modulate GSK-3ß activity without leading to its complete inhibition.

Inhibition of N-acylethanolamine-hydrolyzing acid amidase reduces T cell infiltration in a mouse model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), in which myeloid cells sustain inflammation, take part in priming, differentiation, and reactivation of myelin-specific T cells, and cause direct myelin damage. N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is a proinflammatory enzyme induced by phlogosis and overexpressed in macrophages and microglia of EAE mice. Targeting these cell populations by inhibiting NAAA may be a promising pharmacological strategy to modulate the inflammatory aspect of MS and manage disease progression. To address this goal, we used ARN16186, a small molecule specifically designed and synthesized as a pharmacological tool to inhibit NAAA. We assessed whether enzyme inhibition affected the severity of neurological symptoms and modulated immune cell infiltration into the central nervous system of EAE mice. We found that preventive chronic treatment with ARN16186 was efficacious in slowing disease progression and preserving locomotor activity in EAE mice. Furthermore, NAAA inhibition reduced the number of immune cells infiltrating the spinal cord and modulated the overactivation of NF-kB and STAT3 transcription factors, leading to less expansion of Th17 cells over the course of the disease.

Cytoprotective, anti-inflammatory, and antioxidant properties of high-molecular-weight hyaluronan enriched with red orange extract in human fibroblasts exposed to ultra violet light B irradiation.

Ultraviolet (UV) light exposure is the primary factor responsible for skin photoaging, affecting all the skin layers, mainly through the production of reactive oxygen species (ROS), activation of inflammatory responses, and apoptosis. In keeping with this evidence, exogenous supplementation with dietary antioxidants has been shown to provide photoprotective benefits. Moreover, oral administration of hyaluronic acid (HA) has been proved to reduce the signs of aged skin, such as wrinkles, and increase hydration and elasticity. The combination of different biologically active substances in order to slow down the onset of skin aging could represent a promising preventive strategy against photoaging. In the present study, we investigated the effects of a dietary supplement (IALUTEC® RED), consisting of high-molecular-weight HA (HMW-HA) combined with red orange extract (ROC-Red Orange Complex® ), in human fibroblasts exposed to ultra violet light B-induced oxidative stress. Our study suggests that, in fibroblasts exposed to UVB light, IALUTEC® RED is active in decreasing both the inflammatory response and the generation of ROS, two events that are involved in skin photoaging.

Gambogic acid counteracts mutant p53 stability by inducing autophagy.

Mutant p53 (mutp53) proteins are frequently present at higher levels than the wild-type (wt) protein in tumors, and some of them can acquire oncogenic properties. Consistently, knockdown of mutp53 protein in human cancer cell lines leads to reduced cell proliferation and invasion as well as to an increased sensitivity to some anticancer drugs. Therefore, the exploitation of cellular pathways and/or molecules that promote mutp53 degradation may have a therapeutic interest. Recently, autophagy is emerging as an important pathway involved in the stability of mutp53. In this paper, we explored the autophagic potential of gambogic acid (GA), a molecule that stimulates the degradation of mutp53 and increases the sensitivity of cancer cells to chemotherapeutic agents. We demonstrated that GA may induce mutp53 degradation through autophagy in cancer cells expressing the p53-R280K (MDA-MB-231) and the p53-S241F (DLD1) proteins. The inhibition of autophagy with bafilomycin A1 or chloroquine counteracted mutp53 degradation by GA. However, the autophagy induction and mutp53 degradation affected cell survival and proliferation only at low GA concentrations. At higher GA concentrations, when cells undergo massive apoptosis, autophagy is no longer detectable by immuno-fluorescence analysis. We concluded that autophagy is a relevant pathway for mutp53 degradation in cancer cells but it contributes only partially to GA-induced cell death, in a time and dose-dependent manner.

Cytotoxic activity of Holothuria tubulosa (Echinodermata) coelomocytes.

The immune system of marine invertebrates, in particular that of holothurians, still requires further study. Our research showed that coelomocyte cells contained in the coelomic fluid of the sea cucumber, Holothuria tubulosa, are able to lyse, in vitro, red blood cells in rabbits and sheep. A plaque-forming assay showed spherule cells to be the effector cells, able to release cytotoxic molecules after xenogenic cell contact. The coelomocyte lysate supernatant, analysed by polyacrylamide gel electrophoresis overlay technique, using rabbit and sheep erythrocytes, showed two different haemolytic protein patterns: one calcium dependent and the other calcium independent. The fractions of each pattern were resolved on a polyacrylamide gel and calcium-dependent and independent coelomocyte lysate patterns were compared.

The good, the bad and the ugly: Emys trinacris, Placobdella costata and Haemogregarina stepanowi in Sicily (Testudines, Annelida and Apicomplexa).

Endemic Sicilian pond turtles Emys trinacris Fritz, Fattizzo, Guicking, Tripepi, Pennisi, Lenk, Joger et Wink were examined for the presence of haemogregarine parasites. The presence of haemogregarines, occurring mainly in the microgametocyte stage (13.2 ± 0.12 µm in length and 6.4 ± 0.52 µm in width), was observed in approximately 9% of the sampled E. trinacris. Based on the observed morphology and on the sequencing of nuclear 18S rDNA, we identified the parasite as Haemogregarina stepanowi Danilewsky, 1885. Morphometric study of uninfected and infected red blood cells has shown that H. stepanowi induces different changes in erythrocyte shape depending on the infective stage. The differential count of leukocytes in specimens infected with H. stepanowi showed no significant difference compared with healthy specimens. However, considering the health problems which might be induced by H. stepanowi in the closely related European pond turtle Emys orbicularis (Linneaus), monitoring of the health status of the infected Sicilian populations of E. trinacris is desirable. The restricted distribution of populations of Emys infected with haemogregarines in Sicily is quite puzzling and the possible human-mediated introduction of the parasite in Sicily is briefly discussed.

Second-Generation Non-Covalent NAAA Inhibitors are Protective in a Model of Multiple Sclerosis.

Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are endogenous lipid mediators that suppress inflammation. Their actions are terminated by the intracellular cysteine amidase, N-acylethanolamine acid amidase (NAAA). Even though NAAA may offer a new target for anti-inflammatory therapy, the lipid-like structures and reactive warheads of current NAAA inhibitors limit the use of these agents as oral drugs. A series of novel benzothiazole-piperazine derivatives that inhibit NAAA in a potent and selective manner by a non-covalent mechanism are described. A prototype member of this class (8) displays high oral bioavailability, access to the central nervous system (CNS), and strong activity in a mouse model of multiple sclerosis (MS). This compound exemplifies a second generation of non-covalent NAAA inhibitors that may be useful in the treatment of MS and other chronic CNS disorders.

PRIMA-1 induces autophagy in cancer cells carrying mutant or wild type p53.

PRIMA-1 is a chemical compound identified as a growth suppressor of tumor cells expressing mutant p53. We previously found that in the MDA-MB-231 cell line expressing high level of the mutant p53-R280K protein, PRIMA-1 induced p53 ubiquitination and degradation associated to cell death. In this study, we investigated the ability of PRIMA-1 to induce autophagy in cancer cells. In MDA-MB-231 and HCT116 cells, expressing mutant or wild type p53, respectively, autophagy occurred following exposure to PRIMA-1, as shown by acridine orange staining, anti-LC3 immunofluorescence and immunoblots, as well as by electron microscopy. Autophagy was triggered also in the derivative cell lines knocked-down for p53, although to a different extent than in the parental cells expressing mutant or wild type p53. In particular, while wild type p53 limited PRIMA-1 induced autophagy, mutant p53 conversely promoted autophagy, thus sustaining cell viability following PRIMA-1 treatment. Therefore, the autophagic potential of PRIMA-1, besides being cell context dependent, could be modulated in a different way by the presence of wild type or mutant p53. Furthermore, since both cell lines lacking p53 were more sensitive to the cytotoxic effect of PRIMA-1 than the parental ones, our findings suggest that a deregulated autophagy may favor cell death induced by this drug.

A novel snRNA-like transcript affects amyloidogenesis and cell cycle progression through perturbation of Fe65L1 (APBB2) alternative splicing.

FE65 proteins constitute a family of adaptors which modulates the processing of amyloid precursor protein and the consequent amyloid ß production. Thus, they have been involved in the complex and partially unknown cascade of reactions at the base of Alzheimer's disease etiology. However, FE65 and FE65-like proteins may be linked to neurodegeneration through the regulation of cell cycle in post-mitotic neurons. In this work we disclose novel molecular mechanisms by which APBB2 can modulate APP processing. We show that APBB2 mRNA splicing, driven by the over-expression of a novel non-coding RNA named 45A, allow the generation of alternative protein forms endowed with differential effects on Aß production, cell cycle control, and DNA damage response. 45A overexpression also favors cell transformation and tumorigenesis leading to a marked increase of malignancy of neuroblastoma cells. Therefore, our results highlight a novel regulatory pathway of considerable interest linking APP processing with cell cycle regulation and DNA-surveillance systems, that may represent a molecular mechanism to induce neurodegeneration in post-mitotic neurons.

Intrapleural minocycline pleurodesis for bilateral pneumothorax due to septic pulmonary embolism: a case report.

Background: Pneumothorax is a rare but serious complication of septic pulmonary embolism (SPE). SPE is a life-threatening disorder wherein infected thrombi bring infarction of the terminal and small caliber parts of the pulmonary vasculature and develop multiple nodular and cavitary lesions. Interventions other than conservative chest tube drainage for pneumothorax due to SPE have rarely been reported. Here, we present a case of bilateral pneumothorax due to SPE treated with intrapleural minocycline pleurodesis. Case Description: A 72-year-old male patient previously diagnosed as esophageal carcinoma developed metachronous bilateral pneumothorax while treated for brain metastases. Based on blood cultures and chest computed tomography images, he was diagnosed with pneumothorax secondary to SPE due to methicillin-susceptible Staphylococcus aureus bacteremia. Bilateral chest tube drainage was instituted. Continuous air leakage was found bilaterally after chest tube placement. He was treated with broad-spectrum antibiotics based on the susceptibility profile and supportive treatment for sepsis. Approximately 3 weeks later, air leakage significantly reduced. We performed intrapleural minocycline pleurodesis bilaterally to prevent the recurrence of pneumothorax; the left side was firstly treated and the right side was treated 2 weeks later. Both chest tubes were successfully removed two days after procedures. Although the patient finally died of brain metastases 1 month after pleurodesis, he never recurred pneumothorax. Conclusions: Intrapleural minocycline pleurodesis may be one of the useful and efficacious options in terms of treating intractable pneumothorax associated with SPE. Intrapleural minocycline pleurodesis could be a consideration for intractable pneumothorax related to SPE.

EEC- and ADULT-associated TP63 mutations exhibit functional heterogeneity toward P63 responsive sequences.

TP63 germ-line mutations are responsible for a group of human ectodermal dysplasia syndromes, underlining the key role of P63 in the development of ectoderm-derived tissues. Here, we report the identification of two TP63 alleles, G134V (p.Gly173Val) and insR155 (p.Thr193_Tyr194insArg), associated to ADULT and EEC syndromes, respectively. These alleles, along with previously identified G134D (p.Gly173Asp) and R204W (p.Arg243Trp), were functionally characterized in yeast, studied in a mammalian cell line and modeled based on the crystal structure of the P63 DNA-binding domain. Although the p.Arg243Trp mutant showed both complete loss of transactivation function and ability to interfere over wild-type P63, the impact of p.Gly173Asp, p.Gly173Val, and p.Thr193_Tyr194insArg varied depending on the response element (RE) tested. Interestingly, p.Gly173Asp and p.Gly173Val mutants were characterized by a severe defect in transactivation along with interfering ability on two DN-P63α-specific REs derived from genes closely related to the clinical manifestations of the TP63-associated syndromes, namely PERP and COL18A1. The modeling of the mutations supported the distinct functional effect of each mutant. The present results highlight the importance of integrating different functional endpoints that take in account the features of P63 proteins' target sequences to examine the impact of TP63 mutations and the associated clinical variability.

Gender differences in the immune system activities of sea urchin Paracentrotus lividus.

In the immune system of vertebrates, gender-specific differences in individual immune competence are well known. In general, females possess more powerful immune response than males. In invertebrates, the situation is much less clear. For this purpose we have chosen to study the immune response of the two sexes of the echinoderm Paracentrotus lividus in pre- and post-spawning phases. The coelomic fluid from the echinoderms contains several coelomocyte types and molecules involved in innate immune defenses. In this article we report that the degree of immune responses in the P. lividus differs according to sex in both pre- and post-spawning phases. We found in all tests that females were more active than males. The results indicate that females possess a significant higher number of immunocytes consisting of phagocytes and uncolored spherulocytes. Since the immunological activity is mainly based on immunocytes, it was not surprising that females possessed the highest values of cytotoxicity and hemolysis activity and showed a greater ability to uptake neutral red and phagocyte yeasts cells, while the average number of ingested particles per active phagocyte was not significantly different. Furthermore, agglutinating activity was more evident in the coelomocyte lysate and coelomic fluid of females than in those of males. Finally we found that the acidic extract of female gonads possessed greater antimicrobial activity than that of male gonads. These results make it very likely that gender differences in the immune response are not restricted to vertebrates; rather, they are a general evolutionary phenomenon.

Successful percutaneous catheter-directed treatment of high-risk pulmonary embolism: a case report.

Background: Pulmonary embolism (PE) is an acute cardiovascular syndrome characterized by high incidence and mortality. The therapy of this condition is based on anticoagulation and hemodynamic support, but in case of high-risk mortality, the European Society of Cardiology (ESC) guidelines recommend systemic thrombolytic therapy and surgical embolectomy if thrombolysis is contraindicated or has failed; nowadays several percutaneous catheter-directed treatments for local thrombolysis or mechanical embolectomy are available, but they have IIa class of recommendation, because of lack of robust scientific evidence favoring their use. Case Description: We described a case of high-risk PE treated with a novel percutaneous system for mechanical embolectomy, which consists of a large aspiration catheter that was advanced in the pulmonary artery, capturing and removing a vast thrombus, of 15 centimeters in length. This therapeutic strategy avoided the risk of hemorrhagic complications related to systemic thrombolysis, exiting in the achievement of fast patient hemodynamic stabilization and symptoms resolution, without complications. Computed tomography (CT) pulmonary angiography after 10 days from the intervention revealed the complete resolution of pulmonary artery filling defects, and the patient was discharged asymptomatic. Conclusions: Percutaneous catheter-directed treatments represent an effective alternative therapy for PE, but further studies are needed to demonstrate safety and superiority over the actually recommended therapy.

Rationale and design of the sirolimus eluting balloon utilization for treatment of vasculogenic erectile dysfunction: the SUASION Registry.

BACKGROUND: Conventional angioplasty balloon catheter, drug coated balloon (DCB), or angioplasty with drug eluting stent (DES) have been used on the percutaneous treatment of erectile dysfunction (ED). Currently available DCBs are paclitaxel eluting balloon (PEB), very recently, sirolimus eluting balloon (SEB). Although endovascular revascularization with balloon resulted in improvement of ED, there have been no prior reports on the feasibility of SEB treatment for ED. METHODS: We present an observational, retrospective-prospective multicentre registry in patients evaluating the use of SEB for the treatment of de novo stenosis in native internal pudendal arteries. We will include 100 patients affected by vasculogenic ED non responder to PDE5i with up to two lesions requiring treatment. ED patients should present a IIEF-5 Score<15, positive dynamic doppler (PSV <25 cm/s) and/or evidence at basal CT angiography. At 30 days, 180, 240, and 365 days following the index procedure, IIEF-5 score will be assessed, and medication regimen and adverse event monitoring will be assessed. At 8 months a dynamic Doppler will be performed. Patients will be followed up for 2 years. The primary endpoints are the Delta IIEF-5 Score and a Delta PSV between basal and 8 months follow-up. The secondary endpoint is the incidence of major adverse event (MAE), binary restenosis and late loss in patients who will repeat control angiography if clinically indicated. CONCLUSIONS: Considering the limitations and safety concerns of PEB, POBA and DES used so far in ED clinical investigations, we hypothesize that sirolimus nanocarriers-coated balloon can potentially be an improved next-generation treatment for ED patients.

Does Bacillus thuringiensis Affect the Stress and Immune Responses of Rhynchophorus ferrugineus Larvae, Females, and Males in the Same Way?

Bacillus thuringiensis (Bt) is considered a potentially useful entomopathogen against red palm weevil (RPW) Rhynchophorus ferrugineus. We compared the effects of Bt on mature larvae, females, and males. The pathogenicity of Bt was evaluated, estimating: Median Lethal Dose (LD50), Median Lethal Time (LT50), Total Hemocyte Count (THC), and Differential Hemocyte Counts (DHC), and the expression of the stress protein Heat Shock Protein 70 (Hsp 70) in hemocytes and the brain. Mortality exhibited a positive trend with the dosage and duration of exposure to Bt. Larvae were more susceptible than adults, and the LD50 of females was almost double the value of that of the larvae. LT50 value was higher for females than for males and larvae. Treatment with sub-lethal doses of Bt induced a decrease in THC in larvae, females, and males. In treated larvae, plasmatocytes decreased, while oenocytes and spherulocytes increased. In treated females, all types of hemocytes decreased, while in males the number of plasmatocytes decreased and granulocytes increased. We also registered the stress response directly on hemocytes showing that, already at 3 h after eating Bt, the expression of the stress protein Hsp 70 was modulated. This effect was also observed in brain tissue at 6 h after treatment. The results confirm that Bt treatment induces a pathogenic state in larvae and adults of both sexes, with effects after only a few hours from ingestion; however, the effects are different in magnitude and in type of target.

ARN25068, a versatile starting point towards triple GSK-3ß/FYN/DYRK1A inhibitors to tackle tau-related neurological disorders.

The human kinome plays a crucial role in several pathways. Its dysregulation has been linked to diverse central nervous system (CNS)-related disorders with a drastic impact on the aging population. Among them, tauopathies, such as Alzheimer's Disease (AD) and Frontotemporal Lobar degeneration (FTLD-tau), are neurodegenerative disorders pathologically defined by the presence of hyperphosphorylated tau-positive intracellular inclusions known as neurofibrillary tangles (NFTs). Compelling evidence has reported the great potential of the simultaneous modulation of multiple protein kinases (PKs) involved in abnormal tau phosphorylation through a concerted pharmacological approach to achieve a superior therapeutic effect relative to classic "one target, one drug" approaches. Here, we report on the identification and characterization of ARN25068 (4), a low nanomolar and well-balanced dual GSK-3ß and FYN inhibitor, which also shows inhibitory activity against DYRK1A, an emerging target in AD and tauopathies. Computational and X-Ray studies highlight compound 4's typical H-bonding pattern of ATP-competitive inhibitors at the binding sites of all three PKs. In a tau phosphorylation assay on Tau0N4R-TM-tGFP U2OS cell line, 4 reduces the extent of tau phosphorylation, promoting tau-stabilized microtubule bundles. In conclusion, this compound emerges as a promising prototype for further SAR explorations to develop potent and well-balanced triple GSK-3ß/FYN/DYRK1A inhibitors to tackle tau hyperphosphorylation.

Plaque Prolapse after Stent Implantation in Ectasiant Coronary Artery Atherosclerotic Disease and Large Plaque Burden.

Plaque prolapse (PP) is commonly defined as tissue extrusion through the stent strut. It is not a rare event, frequently detected by intravascular ultrasound, and it is associated with stent thrombosis and adverse outcomes. We present a case of PP after stenting of the left anterior descending coronary artery.

Giant pericardial mass case report: role of echocardiography.

Cardiac masses are rare and they are distinguished in tumors and non-tumoral masses. Primary pericardial masses are very rare and they are often asymptomatic, even if they can present with sudden cardiac death. The diagnosis of these masses is often accidental and they are generally identified with echocardiography; their characterization is usually performed by cardiac magnetic resonance imaging (MRI) but the definitive diagnosis is achieved by tissue biopsy. We described a case of primary pericardial mass in an old patient with history of hypertension, which presented at our hospital with dyspnea and low-extremity edema. The echocardiography described a giant iso/hypoechoic pericardial mass that extended on anterior, posterior and lateral walls of left ventricle and atrium, associated with pericardial effusion without hemodynamic compromise. We discovered that the mass was identified twenty years ago on a chest-computed tomography (CT). Even if we do not manage in performing a cardiac MRI, from echo characteristics we supposed that the mass was a lipoma. Lipomas are benign tumors that can develop from pericardium and they have slow growth so they can be asymptomatic for several years. Their excision is important because they may be responsible for pericardial tamponade or heart failure. Echocardiography is an economic non-invasive exam and it is helpful in differential diagnosis, treatment, follow-up and prognosis of this cardiac masses.

Discovery and SAR Evolution of Pyrazole Azabicyclo[3.2.1]octane Sulfonamides as a Novel Class of Non-Covalent N-Acylethanolamine-Hydrolyzing Acid Amidase (NAAA) Inhibitors for Oral Administration.

Inhibition of intracellular N-acylethanolamine-hydrolyzing acid amidase (NAAA) activity is a promising approach to manage the inflammatory response under disabling conditions. In fact, NAAA inhibition preserves endogenous palmitoylethanolamide (PEA) from degradation, thus increasing and prolonging its anti-inflammatory and analgesic efficacy at the inflamed site. In the present work, we report the identification of a potent, systemically available, novel class of NAAA inhibitors, featuring a pyrazole azabicyclo[3.2.1]octane structural core. After an initial screening campaign, a careful structure-activity relationship study led to the discovery of endo-ethoxymethyl-pyrazinyloxy-8-azabicyclo[3.2.1]octane-pyrazole sulfonamide 50 (ARN19689), which was found to inhibit human NAAA in the low nanomolar range (IC50 = 0.042 µM) with a non-covalent mechanism of action. In light of its favorable biochemical, in vitro and in vivo drug-like profile, sulfonamide 50 could be regarded as a promising pharmacological tool to be further investigated in the field of inflammatory conditions.

PRIMA-1 cytotoxicity correlates with nucleolar localization and degradation of mutant p53 in breast cancer cells.

PRIMA-1 has been identified as a compound that restores the transactivation function to mutant p53 and induces apoptosis in cells expressing mutant p53. Studies on subcellular distribution of the mutant p53 protein upon treatment with PRIMA-1Met, a methylated form of PRIMA-1, have suggested that redistribution of mutant p53 to nucleoli may play a role in PRIMA-1 induced apoptosis. Here, we specifically investigated the influence of PRIMA-1 on cellular localization of mutated p53-R280K endogenously expressed in tumour cells. By using immunofluorescence staining, we found a strong nucleolar redistribution of mutant p53 following PRIMA-1 treatment. This subcellular localization was associated to p53 degradation via ubiquitylation. When cells were treated with adriamycin, neither nucleolar redistribution nor mutant p53 down modulation and degradation were observed. Interestingly, cells where p53-R280K was silenced were more sensitive to PRIMA-1 than the parental ones. These results indicate that in some cellular context, the cell sensitivity to PRIMA-1 could depend on the abolition of a gain-of-function activity of the mutated p53, through a protein degradation pathway specifically induced by this compound.