Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative,NAPA, and the IKKα Kinase

The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose (NAPA), was shown to inhibit the kinase activity of IKKα, one of the two catalytic subunits of IKK complex, decreasing the inflammatory status in osteoarthritis chondrocytes. In the present work we have investigated the inhibition mechanism of IKKα by NAPA by combining computational simulations, in vitro assays and Mass Spectrometry (MS) technique. The kinase in vitro assay was conducted using a recombinant IKKα and IKKtide, a 20 amino acid peptide substrate derived from IkBα kinase protein and containing the serine residues Ser32 and Ser36. Phosphorylated peptide production was measured by Ultra Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS), and the atomic interaction between IKKα and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKKα kinase activity with an IC₅₀ of 0.5 mM, to decrease the K_m value from 0.337 mM to 0.402 mM and the V_max from 0.0257 mM·min−1 to 0.0076 mM·min−1. The computational analyses indicate the region between the KD, ULD and SDD domains of IKKα as the optimal binding site explored by NAPA. Biochemical data indicate that there is a non-significant difference between K_m and K_i whereas there is a statistically significant difference between the two V_max values. This evidence, combined with computational results, consistently indicates that the inhibition is non-competitive, and that the NAPA binding site is different than that of ATP or IKKtide.     

NPM1-Mutated Myeloid Neoplasms with <20% Blasts: A Really Distinct Clinico-Pathologic Entity?

Nucleophosmin (NPM1) gene mutations rarely occur in non-acute myeloid neoplasms (MNs) with <20% blasts. Among nearly 10,000 patients investigated so far, molecular analyses documented NPM1 mutations in around 2% of myelodysplastic syndrome (MDS) cases, mainly belonging to MDS with excess of blasts, and 3% of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) cases, prevalently classified as chronic myelomonocytic leukemia. These uncommon malignancies are associated with an aggressive clinical course, relatively rapid progression to overt acute myeloid leukemia (AML) and poor survival outcomes, raising controversies on their classification as distinct clinico-pathologic entities. Furthermore, fit patients with NPM1-mutated MNs with <20% blasts could benefit most from upfront intensive chemotherapy for AML rather than from moderate intensity MDS-directed therapies, although no firm conclusion can currently be drawn on best therapeutic approaches, due to the limited available data, obtained from small and mainly retrospective series. Caution is also suggested in definitely diagnosing NPM1-mutated MNs with blast count <20%, since NPM1-mutated AML cases frequently present dysplastic features and multilineage bone marrow cells showing abnormal cytoplasmic NPM1 protein delocalization by immunohistochemical staining, therefore belonging to NPM1-mutated clone regardless of blast morphology. Further prospective studies are warranted to definitely assess whether NPM1 mutations may become sufficient to diagnose AML, irrespective of blast percentage.     

Breadmaking performance and technological characteristic of gluten-free bread with inulin supplemented with calcium salts

The fortification of gluten-free bread containing inulin with different organic and non-organic calcium sources was investigated. Calcium lactate, calcium citrate, calcium chloride and calcium carbonate were used as calcium sources. Gluten-free bread composed of corn starch, potato starch, salt, yeast, pectin, sugar and sunflower oil was used as a reference. The calcium salts were supplemented to the gluten-free formula to provide equal content of elementary calcium (Ca⁺²). The Mixolab^? device was used to analyse the behaviour of gluten-free dough subjected to a dual mechanical shear stress and temperature constraint. Calcium salts significantly modified the dough behaviour during heating and cooling. The addition of calcium carbonate and calcium citrate provoked an increase in dough consistency during heating and cooling compared with the other salt-enriched samples. The specific volume and texture parameters of gluten-free breads varied with the calcium salt used, but calcium carbonate and calcium citrate showed improved values. The higher calcium content of the enriched breads, compared with the control, confirmed the fortification. Sensory evaluation of the calcium-fortified breads confirmed that calcium carbonate followed by calcium citrate was the most recommended salt for obtaining calcium fortification of gluten-free breads.     

Source proximity and outdoor-residential VOC concentrations: results from the RIOPA study

Ambient volatile organic compound concentrations outside residences were measured in Elizabeth, New Jersey as part of the Relationship of Indoor, Outdoor, and Personal A:r (RIOPA) study to assess the influence of proximity of the residences to known ambient emissions sources. The closest distances between the outdoor samplers and emission sources were determined using Geographic Information Systems (GIS)techniques. Multiple regression models were developed for residential ambient concentrations of aromatic hydrocarbons (BTEX), methyl tert butyl ether (MTBE), and tetrachloroethylene (PCE). The natural log transformed ambient concentrations of BTEX were inversely associated with distances to major roadways with high traffic densities and gasoline stations, atmospheric stability, temperature, and wind speed. Ambient MTBE levels were associated with inverse distance to gas stations and interstate highways. Residential ambient PCE concentration was inversely associated with distance to dry cleaning facilities, atmospheric stability, temperature, wind speed, and relative humidity. The linear regression models that include proximity to emission sources and meteorological variables explained 16-45% of the overall variation of ambient residential VOC concentrations. Meteorological conditions, especially atmospheric stability and temperature, explained 60-90% of the total variation in the regression models. The residential ambient air concentrations were 1.5-4 times higher than the urban background levels outside homes very close (<50 m) to ambient emission sources where approximately 7% of the population live. However, the relative increase of risk for disease is small and variations in air concentration in the background urban atmosphere are greater than those from the proximity to roadways.     

Highly Regio‐ and Diastereoselective Palladium‐Catalyzed Allylic Substitution. Synthesis of 3‐(2‐Aminobutylidene)‐ 4‐arylazetidin‐2‐ones

The palladium‐catalyzed benzylamine attack to a particular allylic moiety, the 3‐alkenyl‐3‐bromoazetidin‐2‐one is herein reported. This reaction shows interesting mechanistic aspects and allows us to introduce in one step and under high regio‐ and stereocontrol the amino function in the C3 side chain of non‐conventional β‐lactams, thus offering the opportunity for designing new potential glutamine syntethase inhibitors, such as Tabtoxin analogues.     

Cloning of the Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris

The DEX gene encoding an extracellular dextranase was isolated from the genomic DNA library of Penicillium minioluteum by hybridization using the dextranase cDNA as a probe. Comparison of the gene and cDNA sequences revealed that the DEX gene does not contain introns. Amino acid sequences comparison of P. minioluteum dextranase with other reported dextranases reveals a significant homology (29% identity) with a dextranase from Arthrobacter sp. CB-8. The DEX gene fragment encoding a mature protein of 574 amino acids was expressed in the methylotrophic yeast Pichia pastoris by using the SUC2 gene signal sequence from Saccharomyces cerevisiae under control of the alcohol oxidase-1 (AOX1) promoter. Over 3·2g/l of enzymatically active dextranase was secreted into the medium after induction by methanol. The yeast product was indistinguishable from the native enzyme in specific activity and the N-terminus of both proteins were identical.     

Molecular Fingerprint of BMD Patients Lacking a Portion in the Rod Domain of Dystrophin

BMD is characterized by a marked heterogeneity of gene mutations resulting in many abnormal dystrophin proteins with different expression and residual functions. The smaller dystrophin molecules lacking a portion around exon 48 of the rod domain, named the D8 region, are related to milder phenotypes. The study aimed to determine which proteins might contribute to preserving muscle function in these patients. Patients were subdivided, based on the absence or presence of deletions in the D8 region, into two groups, BMD1 and BMD2. Muscle extracts were analyzed by 2-D DIGE, label-free LC-ESI-MS/MS, and Ingenuity pathway analysis (IPA). Increased levels of proteins typical of fast fibers and of proteins involved in the sarcomere reorganization characterize BMD2. IPA of proteomics datasets indicated in BMD2 prevalence of glycolysis and gluconeogenesis and a correct flux through the TCA cycle enabling them to maintain both metabolism and epithelial adherens junction. A 2-D DIGE analysis revealed an increase of acetylated proteoforms of moonlighting proteins aldolase, enolase, and glyceraldehyde-3-phosphate dehydrogenase that can target the nucleus promoting stem cell recruitment and muscle regeneration. In BMD2, immunoblotting indicated higher levels of myogenin and lower levels of PAX7 and SIRT1/2 associated with a set of proteins identified by proteomics as involved in muscle homeostasis maintenance.     

Oxidation Stress as a Mechanism of Aging in Human Erythrocytes: Protective Effect of Quercetin

Aging is a multi-factorial process developing through a complex net of interactions between biological and cellular mechanisms and it involves oxidative stress (OS) as well as protein glycation. The aim of the present work was to verify the protective role of Quercetin (Q), a polyphenolic flavonoid compound, in a d-Galactose (d-Gal)-induced model of aging in human erythrocytes. The anion-exchange capability through the Band 3 protein (B3p) measured by the rate constant of the SO₄²⁻ uptake, thiobarbituric acid reactive substances (TBARS) levels—a marker of lipid peroxidation—total sulfhydryl (-SH) groups, glycated hemoglobin (A1c), and a reduced glutathione/oxidized glutathione (GSH-GSSG) ratio were determined following the exposure of erythrocytes to 100 mM d-Gal for 24 h, with or without pre-incubation with 10 µM Q. The results confirmed that d-Gal activated OS pathways in human erythrocytes, affecting both membrane lipids and proteins, as denoted by increased TBARS levels and decreased total sulfhydryl groups, respectively. In addition, d-Gal led to an acceleration of the rate constant of the SO₄²⁻ uptake through the B3p. Both the alteration of the B3p function and oxidative damage have been improved by pre-treatment with Q, which preferentially ameliorated lipid peroxidation rather than protein oxidation. Moreover, Q prevented glycated A1c formation, while no protective effect on the endogenous antioxidant system (GSH-GSSG) was observed. These findings suggest that the B3p could be a novel potential target of antioxidant treatments to counteract aging-related disturbances. Further studies are needed to confirm the possible role of Q in pharmacological strategies against aging.     

A Metabolic Signature of Hereditary Transthyretin Amyloidosis: A Pilot Study

Hereditary transthyretin amyloidosis is the most common form of hereditary amyloidosis, with an autosomal dominant inheritance and a variable penetrance. ATTRv amyloidosis can present as a progressive, axonal sensory autonomic and motor neuropathy or as an infiltrative cardiomyopathy. The definition of biomarkers for the early diagnosis of ATTRv is particularly important in the current era of emerging treatments. In this sense, metabolomics could be an instrument able to provide metabolic profiles with their related metabolic pathways, and we would propose them as possible fluid biomarkers. The aim of this study is to identify altered metabolites (free fatty acids and amino acids) in subjects with a confirmed pathogenic TTR variant. Out of the studied total free fatty acids and amino acids, the serum values of palmitic acid are significantly lower in the ATTRv patients compared to the recruited healthy subjects. The metabolic remodeling identified in this neurogenetic disorder could be the manifestation of pathophysiological processes of the disease, such as mitochondrial dysfunction and neuroinflammation, and contribute to explaining some of its clinical manifestations.     

Structure,morphology and magnetic properties of Au/Fe3O4 nanocomposites fabricated by a soft aqueous route

Magnetic Fe₃O₄ (magnetite) nanoparticles are synthesized via a chemical precipitation route in different alkaline environments (NH₃ or NaOH) and subsequently functionalized with a (propynylcarbamate)triethoxysilane moiety, with the aim of promoting the nucleation and subsequent stabilization of gold nanoparticles. The propynylcarbamate group is able to capture the gold precursor (HAuCl₄), spontaneously reduce it, and stabilize the resulting Au nanoaggregates. The obtained results show that though the dimensions of the starting magnetite substrate depend on the base used in the preparation, they remain unaltered upon the subsequent modification. Conversely, the average Au nanoparticle dimensions can be conveniently tailored as a function of the base used in Fe₃O₄ preparation and the presence/absence of the organic functionalization. The smallest dimensions (15?nm) are obtained for AuNP supported on propynylcarbamate-functionalized Fe₃O₄ prepared in the presence of ammonia. Magnetization measurements highlight that all the Au/Fe₃O₄ nanocomposites display a superparamagnetic behavior and those obtained using ammonia showed consistently smaller Hc and Mr values (av. values of 7.4?Oe and 0.8?emu/g) than those prepared with sodium hydroxide (av. values of 28?Oe and 2.8?emu/g).