Maurizio Ballico

Osmium(II) CNN pincer complexes as efficient catalysts for both asymmetric transfer and H2 Hydrogenation of Ketones

We describe the isolation of the first CNN pincer osmium complexes [OsX(CNN)P 2 ] to display high catalytic activity and productivity in the reduction of ketones by either TH or HY (TOFand TON up to 10 6 h 1 and 10 5 , respectively). A high enantioselectivity (up to 98% ee ) is possible in both reactions with a remarkably low catalyst loading (0.005–0.002 mol%). To the best of our knowledge, this is the first example of an osmium complex that catalyzes the asymmetric HY of ketones. Evidence for an Os-OR vs. Os-H equilibrium suggests that both these species are involved in the catalytic pathways. Mechanistic studies as well as the preparation of new CNN pincer osmium catalysts are currently underway.

BMP tests of source selected OFMSW to evaluate anaerobic codigestion with sewage sludge

The aim of this study is to characterize different types of source selected organic fraction of municipal solid waste (SS-OFMSW) in order to optimize the upgrade of a sewage sludge anaerobic digestion unit by codigestion. Various SS-OFMSW samples were collected from canteens, supermarkets, restaurants, households, fruit-vegetable markets and bakery shops. The substrates characterization was carried out getting traditional chemical-physical parameters, performing elemental analysis and measuring fundamental anaerobic digestion macromolecular compounds such as carbohydrates, proteins, lipids and volatile fatty acids. Biochemical methane potential (BMP) tests were conducted at mesophilic temperature both on single substrates and in codigestion regime with different substrates mixing ratios. The maximum methane yield was observed for restaurant (675 NmlCH4/gVS) and canteens organic wastes (571 and 645 NmlCH4/gVS). The best codigestion BMP test has highlighted an increase of 47% in methane production respect sewage sludge digestion.

Osmium Pyme Complexes for Fast Hydrogenation and Asymmetric Transfer Hydrogenation of Ketones

The osmium compound trans,cis-[OsCl2(PPh3)2(Pyme)] (1) (Pyme=1-(pyridin-2-yl)methanamine), obtained from [OsCl2(PPh3)3] and Pyme, thermally isomerizes to cis,cis-[OsCl2(PPh3)(2)(Pyme)] (2) in mesitylene at 150 degrees C. Reaction of [OsCl2(PPh3)3] with Ph2P(CH2)(4)PPh2 (dppb) and Pyme in mesitylene (150 degrees C, 4 h) leads to a mixture of trans-[OsCl2(dppb)(Pyme)] (3) and cis-[OsCl2(dppb)(Pyme)] (4) in about an 1:3 molar ratio. The complex trans-[OsCl2(dppb)(Pyet)] (5) (Pyet=2-(pyridin-2-yl)ethanamine) is formed by reaction of [OsCl2(PPh3)3] with dppb and Pyet in toluene at reflux. Compounds 1, 2, 5 and the mixture of isomers 3/4 efficiently catalyze the transfer hydrogenation (TH) of different ketones in refluxing 2-propanol and in the presence of NaOiPr (2.0 mol %). Interestingly, 3/4 has been proven to reduce different ketones (even bulky) by means of TH with a remarkably high turnover frequency (TOF up to 5.7 x 10(5) h(-1)) and at very low loading (0.05-0.001 mol %). The system 3/4 also efficiently catalyzes the hydrogenation of many ketones (H2, 5.0 atm) in ethanol with KOtBu (2.0 mol %) at 70 degrees C (TOF up to 1.5 x 10(4) h(-1)). The in-situ-generated catalysts prepared by the reaction of [OsCl2(PPh3)3] with Josiphos diphosphanes and (+/-)-1-alkyl-substituted Pyme ligands, promote the enantioselective TH of different ketones with 91-96 % ee (ee=enantiomeric excess) and with a TOF of up to 1.9 x 10(4) h(-1) at 60 degrees C.

Antiproliferative activity of a triplex-forming oligonucleotide recognizing a Ki- ras polypurine/polypyrimidine motif correlates with protein binding

The Ki-ras gene is frequently mutated and/or overexpressed in human cancer. Since it is suspected to play a key role in the pathogenesis of many tumors, there is interest to search for strategies aiming at the specific inhibition of this oncogene. In this paper, we investigated the capacity of a 20 mer G-rich oligonucleotide (ODN20) conjugated to high molecular weight monomethoxy polyethylene glycol (MPEG) to inhibit the expression of the Ki-ras gene and the proliferation of pancreatic cancer cells. The conjugate, MPEG ODN20, was designed to form a triplex with a critical pur/pyr sequence located in the promoter of the Ki-ras gene. To make the conjugate resistant to endogenous and exogenous nucleases, five phosphorothioate linkages were introduced in its backbone. Confocal microscopy and FACS experiments showed that MPEG ODN20 had a higher capacity to penetrate the cell membranes and accumulate in the nucleus of Panc-1 cells than ODN20. Incubation of Panc-1 cells with MPEG ODN20 reduced specifically the levels of Ki-ras mRNA and RAS protein p21RAS. A single-dose administration of MPEG ODN20 was sufficient to inhibit cell proliferation by about 50% compared with control. By contrast, the antiproliferative activity of the unconjugated ODN20 analog was found to be not significant. Band-shift and footprinting experiments showed that MPEG ODN20 formed a weak triplex (Kd ∼1.5 μM at 37°C, 50 mM Tris-acetate, pH 7.4, 10 mM NaCl, 10 mM MgCl2, 5 mM spermidine) with the Ki-ras pyr/pur motif, suggesting that its bioactivity can hardly be mediated by a triplex-based mechanism. Here, we provide evidence that, in vitro, ODN20 and MPEG ODN20 competitively inhibit the binding to the Ki-ras pur/pyr motif of a nuclear protein, suggesting that the activity of MPEG ODN20 occurs with an aptameric mechanism. The biological implications of this study are discussed.

Altered Metabolic Profile in Congenital Lung Lesions Revealed by 1H Nuclear Magnetic Resonance Spectroscopy

Congenital lung lesions are highly complex with respect to pathogenesis and treatment. Large-scale analytical methods, like metabolomics, are now available to identify biomarkers of pathological phenotypes and to facilitate clinical management. Nuclear magnetic resonance (NMR) is a unique tool for translational research, as in vitro results can be potentially translated into in vivo magnetic resonance protocols. Three surgical biopsies, from congenital lung malformations, were analyzed in comparison with one control sample. Extracted hydrophilic metabolites were submitted to high resolution 1H NMR spectroscopy and the relative concentration of 12 metabolites was estimated. In addition, two-dimensional NMR measurements were performed to complement the results obtained from standard monodimensional experiments. This is one of the first reports of in vitro metabolic profiling of congenital lung malformation. Preliminary data on a small set of samples highlights some altered metabolic ratios, dealing with the glucose conversion to lactate, to the relative concentration of phosphatidylcholine precursors, and to the presence of myoinositol. Interestingly some relations between congenital lung lesions and cancer metabolic alterations are found.

New Syntheses of Branched, Multifunctional High-Molecular Weight Poly(ethylene glycol)s or (MultiPEG)s

A new set of branched high-molecular weight multifunctional poly(ethylene glycol) (MultiPEG) derivatives was obtained from smaller commercial diOH-PEGs (M.W. = 2000 and 6000 Da) which were selectively monoprotected, properly activated at the residual OH-functions and treated with 1,3-diamino-2-propanol and 2-amino-1,3-propandiol as polyfunctional linkers. They were purified by molecular exclusion chromatography or extensive dialysis and characterized by GPC and 1 H-NMR. The final polymeric derivatives are characterized by a higher number of terminal reacting moieties (up to 6 times) than that found in linear, commercial PEGs of same molecular size. This increased loading capability can be applied to improve their use as drug carriers and soluble synthetic supports.

A specific nanobody prevents amyloidogenesis of D76N β2-microglobulin in vitro and modifies its tissue distribution in vivo

Systemic amyloidosis is caused by misfolding and aggregation of globular proteins in vivo for which effective treatments are urgently needed. Inhibition of protein self-aggregation represents an attractive therapeutic strategy. Studies on the amyloidogenic variant of β2-microglobulin, D76N, causing hereditary systemic amyloidosis, have become particularly relevant since fibrils are formed in vitro in physiologically relevant conditions. Here we compare the potency of two previously described inhibitors of wild type β2-microglobulin fibrillogenesis, doxycycline and single domain antibodies (nanobodies). The β2-microglobulin -binding nanobody, Nb24, more potently inhibits D76N β2-microglobulin fibrillogenesis than doxycycline with complete abrogation of fibril formation. In β2-microglobulin knock out mice, the D76N β2-microglobulin/ Nb24 pre-formed complex, is cleared from the circulation at the same rate as the uncomplexed protein; however, the analysis of tissue distribution reveals that the interaction with the antibody reduces the concentration of the variant protein in the heart but does not modify the tissue distribution of wild type β2-microglobulin. These findings strongly support the potential therapeutic use of this antibody in the treatment of systemic amyloidosis.

Functional expression of aryl hydrocarbon receptor on mast cells populating human endometriotic tissues

Endometriosis is an inflammatory disease characterized by the presence of ectopic endometrial tissue outside the uterus. A diffuse infiltration of mast cells (MCs) is observed throughout endometriotic lesions, but little is known about how these cells contribute to the network of molecules that modulate the growth of ectopic endometrial implants and promote endometriosis-associated inflammation. The aryl hydrocarbon receptor (AhR), a transcription factor known to respond to environmental toxins and endogenous compounds, is present in MCs. In response to AhR activation, MCs produce IL-17 and reactive oxygen species, highlighting the potential impact of AhR ligands on inflammation via MCs. Here, we investigated the possibility that endometrial MCs promote an inflammatory microenvironment by sensing AhR ligands, thus sustaining endometriosis development. Using human endometriotic tissue (ET) samples, we performed the following experiments: (i) examined the cytokine expression profile; (ii) counted AhR-expressing MCs; (iii) verified the phenotype of AhR-expressing MCs to establish whether MCs have a tolerogenic (IL-10-positive) or inflammatory (IL-17-positive) phenotype; (iv) measured the presence of AhR ligands (tryptophan-derived kynurenine) and tryptophan-metabolizing enzymes (indoleamine 2,3-dioxygenase 1 (IDO1)); (v) treated ET organ cultures with an AhR antagonist in vitro to measure changes in the cytokine milieu; and (vi) measured the growth of endometrial stromal cells cultured with AhR-activated MC-conditioned medium. We found that ET tissue was conducive to cytokine production, orchestrating chronic inflammation and a population of AhR-expressing MCs that are both IL-17 and IL-10-positive. ET was rich in IDO1 and the AhR-ligand kynurenine compared with control tissue, possibly promoting MC activation through AhR. ET was susceptible to treatment with an AhR antagonist, and endometrial stromal cell growth was improved in the presence of soluble factors released by MCs on AhR activation. These results suggest a new mechanistic role of MCs in the pathogenesis of endometriosis.

Phospholipid Profile of Amniotic Fluid in Ovine Model of Congenital Diaphragmatic Hernia (CDH): The Effect of Fetal Tracheal Occlusion

Fetal endoscopic tracheal occlusion has been proposed as a prenatal intervention to ameliorate congenital diaphragmatic hernia (CDH) prognosis. Tracheal occlusion (TO) prevents pulmonary fluid egress, leading to tissue expansion, reversal of lung hypoplasia, and potential maturation. Fetal lung maturity strongly correlates with amniotic fluid (AF) phospholipidic composition. In this preliminary study, we characterized the AF phospholipidic profile in CDH-induced, TO-treated, and healthy fetal lambs to define the prenatal treatment benefits of TO on lung maturity. CDH induction was performed at 70 days of gestation, TO was carried out at 102 days of gestation, and caesarean section was carried out at 136 days of gestation. AF samples, taken at 102-136 days of gestation, were evaluated using mass spectrometry. The analysis focused on phosphatidylcholines (PCs) and sphingomyelins (SMs). The most abundant phosphatidylcholine species retrieved in healthy AF was POPC [PC(18:1/16:0)], while the level of DPPC [PC(16:0/16:0)] was extremely low at both gestational ages. CDH induction caused a decrease in POPC and many other PCs. A substantial return of some PCs, in particular POPC, PC(34:2) and PC(18:0/16:0), to a more physiological level was prompted by TO. SMs were unaltered. The AF phospholipidic profile could provide prenatal prognostic markers of CDH and possible indices of lung maturation after fetal treatment.

Congenital pulmonary malformations: metabolomic profile of lung phenotype in infants

Abstract Background: The main hydrosoluble metabolites in three different human congenital pulmonary malformations are described by nuclear magnetic resonance (NMR) spectroscopy. Methods: Bronchogenic cyst (BC), congenital lobar emphysema (CLE) and intrapulmonary sequestration (IPS), were analyzed with respect to a control sample. The extracted metabolites were submitted to high-resolution 1H NMR-spectroscopy. Results: Congenital lung malformations showed free choline, phosphocoline and myoinositol high levels. IPS and CLE were found increased in lactic acid/glucose ratio. Lactic acid and glucose values resulted to be more elevated in control sample. Conclusions: Congenital lung lesions showed different metabolomic profiles useful for early diagnosis.