Effect of CB2 Stimulation on Gene Expression in Pediatric B-Acute Lymphoblastic Leukemia: New Possible Targets

Acute lymphoblastic leukemia type B (B-ALL) is the most common kind of pediatric leukemia, characterized by the clonal proliferation of type B lymphoid stem cells. Important progress in ALL treatments led to improvements in long-term survival; nevertheless, many adverse long-term consequences still concern the medical community. Molecular and cellular target therapies, together with immunotherapy, are promising strategies to overcome these concerns. Cannabinoids, enzymes involved in their metabolism, and cannabinoid receptors type 1 (CB1) and type 2 (CB2) constitute the endocannabinoid system, involved in inflammation, immune response, and cancer. CB2 receptor stimulation exerts anti-proliferative and anti-invasive effects in many tumors. In this study, we evaluated the effects of CB2 stimulation on B-ALL cell lines, SUP-B15, by RNA sequencing, Western blotting, and ELISA. We observe a lower expression of CB2 in SUP-B15 cells compared to lymphocytes from healthy subjects, hypothesizing its involvement in B-ALL pathogenesis. CB2 stimulation reduces the expression of CD9, SEC61G, TBX21, and TMSB4X genes involved in tumor growth and progression, and also negatively affects downstream intracellular pathways. Our findings suggest an antitumor role of CB2 stimulation in B-ALL, and highlight a functional correlation between CB2 receptors and specific anti-tumoral pathways, even though further investigations are needed.     

Enhanced deposition and bioaccumulation of mercury in Antarctic terrestrial ecosystems facing a coastal polynya

Mercury emitted by anthropogenic and natural sources occurs in the atmosphere mostly in the gaseous elemental form, which has a long lifetime in tropical and temperate regions. Once deposited in terrestrial and aquatic ecosystems the metal is partly re-emitted into the air, thus assuming the characteristics of global pollutants such as persistent volatile chemicals. In polar regions, during and after the sunrise, the photochemically driven oxidation of gaseous Hg by reactive halogens may result in areas of greatly enhanced Hg deposition. Mercury concentrations in soils, lichens, and mosses collected in a stretch between 74 degrees 30' S and 76 degrees 00' S, in ice-free coastal areas of Victoria Land facing the Terra Nova Bay coastal polynya, were higher than typical Antarctic baselines. The finding of enhanced Hg bioaccumulation in Antarctic terrestrial ecosystems facing a coastal polynya strongly supports recent speculations on the role of ice crystals ("frost flowers") growing in polynyas as a dominant source of sea salt aerosols and bromine compounds, which are involved in springtime mercury depletion events (MDEs). These results raise concern aboutthe possible environmental effects of changes in regional climate and sea ice coverage, and on the possible role of Antarctica as a sink in the mercury cycle.     

At Embryo Implantation Site IL-35 Secreted by Trophoblast,Polarizing T Cells towards IL-35+ IL-10+ IL-4+ Th2-Type Cells,Could Favour Fetal Allograft Tolerance and Pregnancy Success

We investigated the role of rhIL-35, at low concentrations compatible with those produced by human trophoblast cells (less than 1 ng/mL), on human T helper (Th) cell functions and the presence of decidual IL-35-producing Th cells in human pregnancy. We found that human trophoblast cells produced IL-35 but not IL-4 or IL-10. RhIL-35, at concentrations produced by human trophoblasts, polarized T cells towards IL-35+, IL-10+, IL-4+ Th2-type cells and to Foxp3+ EBI3+ p35+ T reg cells producing IL-35 but not IL-10 and IL-4. Moreover, rhIL-35 at low concentrations did not suppress the proliferation of Th cells but stimulated IL-4 and IL-10 production by established Th clones. In particular, Th1-type clones acquired the capacity to produce IL-4. In addition, purified human trophoblast cell supernatants containing IL-35 upregulated IL-4 and IL-10 production by Th clones. Finally, IL-35+, IL-10+, IL-4+ Th2-type cells, which were found to be induced by low concentrations of IL-35 compatible with those produced by human trophoblasts, are exclusively present in the decidua of a successful pregnancy and at the embryo implantation site, suggesting their stringent dependence on trophoblast cells. Thus, the proximity of Th cells to IL-35-producing trophoblasts could be the determining factor for the differentiation of IL-35+, IL-10+, IL-4+ Th2-type cells that are crucial for human pregnancy success.     

Percutaneous closure of congenital and iatrogenic coronary arterial fistulas

Coronary arteriovenous fistulas (CAF) are the most common hemodinamically significant congenital coronary anomalies. Surgery has been the only therapeutic option for ages. We describe three cases of percutaneous occlusion of CAF, congenital and iatrogenic, that we treated with different devices, to fit their different anatomic and functional characteristics. Case 1). Male patient (pt) 20 years old, asymptomatic, affected with CAF between the right coronary artery and the right ventricle, with aneurysmatic vessel dilatation and occlusion of the posterolateral branches. CAF has been occluded with a detachable, valvulated latex balloon, wedged into the proximal neck of the aneurysm. Case 2). Female pt 63 years old, who was symptomatic for exertion angina, affected with multiple CAF which originated from proximal and distal circumflex artery, proximal left anterior descending artery (LAD), all of which flowed into the left inferior lobar pulmonary artery. The fistulas have been occluded with steel and tungsten coils. Case 3). Male pt 62 years old, who underwent orthotopic cardiac transplantation in 1990 for dilated cardiomyopathy. Coronary angiogram at one year was normal, but subsequently a multilocular CAF between LAD in the middle portion and the right ventricle became evident. During angiographic follow-up an increase of the size of the fistula was observed, together with a reduction of that of distal LAD. For this reason a percutaneous occlusion with multiple tungsten coil has been performed. The three procedures have had a favorable outcome and we did not observe any acute or late complications; clinical and angiographic follow-up confirmed this satisfactory results at six months. Based on the data of the literature and on this experience, we conclude that percutaneous occlusion is the first line therapy of CAF and that the different devices can be tailored to meet different anatomic and functional characteristics.     

A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels

Connexin (Cx) hemichannels (HCs) are large pore hexameric structures that allow the exchange of ions, metabolites and a variety of other molecules between the cell cytoplasm and extracellular milieu. HC inhibitors are attracting growing interest as drug candidates because deregulated fluxes through HCs have been implicated in a plethora of genetic conditions and other diseases. HC activity has been mainly investigated by electrophysiological methods and/or using HC-permeable dye uptake measurements. Here, we present an all-optical assay based on fluorometric measurements of ionized calcium (Ca²⁺) uptake with a Ca²⁺-selective genetically encoded indicator (GCaMP6s) that permits the optical tracking of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) changes with high sensitivity. We exemplify use of the assay in stable pools of HaCaT cells overexpressing human Cx26, Cx46, or the pathological mutant Cx26G45E, under control of a tetracycline (Tet) responsive element (TRE) promoter (Tet-on). We demonstrate the usefulness of the assay for the characterization of new monoclonal antibodies (mAbs) targeting the extracellular domain of the HCs. Although we developed the assay on a spinning disk confocal fluorescence microscope, the same methodology can be extended seamlessly to high-throughput high-content platforms to screen other kinds of inhibitors and/or to probe HCs expressed in primary cells and microtissues.     

Inhibition of Bcr-Abl phosphorylation and induction of apoptosis by pyrazolo[3,4-d]pyrimidines in human leukemia cells

A series of pyrazolo[3,4-d]pyrimidines, previously found to be Src inhibitors, was tested for their ability to inhibit proliferation of three Bcr-Abl-positive human leukemia cell lines (K-562, KU-812, and MEG-01), on the basis of the experimental evidence that various Src inhibitors are also active against Bcr-Abl kinase (the so called dual Src/Abl inhibitors). They reduce Bcr-Abl tyrosine phosphorylation and promote apoptosis of the Bcr-Abl-expressing cells. A cell-free enzymatic assay on isolated c-Abl confirmed that such compounds directly inhibit Abl activity. Finally, molecular modeling simulations were also performed to hypothesize the binding mode of the compounds into the Abl binding site.     

Regio- and stereochemistry of the first insertion step in the 1,3-butadiene polymerization catalyzed by CpTiCl3-MAO

CpTiCl₃-MAO (methylaluminoxane) catalytic system promotes the butadiene polymerization leading to 85% of 4,1-cis polymer. The generally well accepted homopolymerization mechanism involves a diene insertion on a π-allylic terminal growing chain. In α-olefin-diene copolymerizations, diene monomer can also insert on a σ carbon-metal bond.In order to simulate an insertion of a conjugated diene on a σ chain, experimental and theoretical studies, relative to the first insertion step, are reported. Unexpectedly 56% of 1,2-primary and 44% of 4,1-trans first inserted units were obtained, by using as catalyst CpTiCl₃/MAO/Al(¹³CH₃)₃. The experimental results were rationalized by DFT (Density Functional Theory) calculations, by including a solvent molecule coordinated to the catalytic site during the first insertion step.     

Scalaradial, a dialdehyde-containing marine metabolite that causes an unexpected noncovalent PLA2 Inactivation

Several marine terpenoids that contain at least one reactive aldehyde group, such as manoalide and its congeners, possess interesting anti-inflammatory activities that are mediated by the covalent inactivation of secretory phospholipase A(2) (sPLA(2)). Scalaradial, a 1,4-dialdehyde marine terpenoid that was isolated from the sponge Cacospongia mollior, is endowed with a relevant anti-inflammatory profile, both in vitro and in vivo, through selective sPLA(2) inhibition. Due to its peculiar dialdehyde structural feature, it has been proposed that scalaradial exerts its enzymatic inactivation by means of an irreversible covalent modification of its target. In the context of our on-going research on anti-PLA(2) natural products and their interaction at a molecular level, we studied scalaradial in an attempt to shed more light on the molecular mechanism of its PLA(2) inhibition. A detailed analysis of the reaction profile between scalaradial and bee venom PLA(2), a model sPLA(2) that shares a high structural homology with the human synovial enzyme, was performed by a combination of spectroscopic techniques, chemical reactions (selective modifications, biomimetic reactions), and classical protein chemistry (such as proteolytic digestion, HPLC and mass spectrometry), along with molecular modeling studies. Unexpectedly, our data clearly indicated the noncovalent forces to be the leading event in the PLA(2) inactivation process; thus, the covalent modification of the enzyme emerges as only a minor side event in the ligand-enzyme interaction. The overall picture might be useful in the design of SLD analogues as new potential anti-inflammatory compounds that target sPLA(2) enzymes.     

The molecular mechanism of bee venom phospholipase A2 inactivation by bolinaquinone

The molecular basis of the inactivation of bee venom PLA2 by the marine natural product bolinaquinone (BLQ) was studied by several spectral techniques (CD, fluorescence, and NMR spectroscopy, mass spectrometry), biomimetic reactions, and molecular modeling. Our data suggest competitive inhibition based on a BLQ-PLA2 noncovalent molecular recognition. However, BLQ is also able to react selectively with Lys133 through conjugate addition followed by a beta elimination. The biological implications of both the covalent and noncovalent molecular events are discussed.     

Mechanically Induced Zone Darkening of Alumina/Ceria-Stabrlized Zirconia Composites

Alumina/ceria-stabilized zirconia bars tested in four-point bending exhibited deformation bands on the tensile side due to localized tetragonal-to-monoclinic transformation. These bands became increasingly darker and were fully visible some weeks after the bending experiment. XPS spectra revealed that the darker regions are characterized by a higher concentration of cerium in the + 3 oxidation state, as opposed to other undeformed regions, in which + 4 is the predominant oxidation state. A numerical calculation established that residual stresses exist in the deformation bands. It is suggested that these stresses facilitate the reduction of cerium with the gradient in stress motivating the diffusion of oxygen vacancies.