Engineered ferritin for lanthanide binding

Ferritin H-homopolymers have been extensively used as nanocarriers for diverse applications in the targeted delivery of drugs and imaging agents, due to their unique ability to bind the transferrin receptor (CD71), highly overexpressed in most tumor cells. In order to incorporate novel fluorescence imaging properties, we have fused a lanthanide binding tag (LBT) to the C-terminal end of mouse H-chain ferritin, HFt. The HFt-LBT possesses one high affinity Terbium binding site per each of the 24 subunits provided by six coordinating aminoacid side chains and a tryptophan residue in its close proximity and is thus endowed with strong FRET sensitization properties. Accordingly, the characteristic Terbium emission band at 544 nm for the HFt-LBT Tb(III) complex was detectable upon excitation of the tag enclosed at two order of magnitude higher intensity with respect to the wtHFt protein. X-ray data at 2.9 Å and cryo-EM at 7 Å resolution demonstrated that HFt-LBT is correctly assembled as a 24-mer both in crystal and in solution. On the basis of the intrinsic Tb(III) binding properties of the wt protein, 32 additional Tb(III) binding sites, located within the natural iron binding sites of the protein, were identified besides the 24 Tb(III) ions coordinated to the LBTs. HFt-LBT Tb(III) was demonstrated to be actively uptaken by selected tumor cell lines by confocal microscopy and FACS analysis of their FITC derivatives, although direct fluorescence from Terbium emission could not be singled out with conventional, 295–375 nm, fluorescence excitation

Iridium oxide (IV) nanoparticle-based electrocatalytic detection of PBDE

Polybrominated diphenyl ethers (PBDEs) are a type of flame retardants which are currently banned in EU and USA due their hazardousness for humans and mammals. However, these compounds were highly used during more than 30 years and still persist in the environment since they are resistant to degradation. Herein we present a biosensor for the detection of PBDEs using screen printed carbon electrodes (SPCEs) based on the electrochemical monitoring of water oxidation reaction (WOR) catalyzed by iridium oxide (IV) nanoparticles (IrO NPs). Our assay shows a limit of detection of 21.5 ppb of PBDE in distilled water. We believe that such an IrO NPs-based electrocatalytic sensing system can lead to a rapid, sensitive, low cost and miniaturizable device for the detection of PBDEs

In vivo migration of labeled autologous natural killer cells to liver metastases in patients with colon carcinoma

BACKGROUND: Besides being the effectors of native anti-tumor cytotoxicity, NK cells participate in T-lymphocyte responses by promoting the maturation of dendritic cells (DC). Adherent NK (A-NK) cells constitute a subset of IL-2-stimulated NK cells which show increased expression of integrins and the ability to adhere to solid surface and to migrate, infiltrate, and destroy cancer. A critical issue in therapy of metastatic disease is the optimization of NK cell migration to tumor tissues and their persistence therein. This study compares localization to liver metastases of autologous A-NK cells administered via the systemic (intravenous, i.v.) versus locoregional (intraarterial, i.a.) routes. PATIENTS AND METHODS: A-NK cells expanded ex-vivo with IL-2 and labeled with (111)In-oxine were injected i.a. in the liver of three colon carcinoma patients. After 30 days, each patient had a new preparation of (111)In-A-NK cells injected i.v. Migration of these cells to various organs was evaluated by SPET and their differential localization to normal and neoplastic liver was demonstrated after i.v. injection of (99m)Tc-phytate. RESULTS: A-NK cells expressed a donor-dependent CD56(+)CD16(+)CD3(- )(NK) or CD56(+)CD16(+)CD3(+ )(NKT) phenotype. When injected i.v., these cells localized to the lung before being visible in the spleen and liver. By contrast, localization of i.a. injected A-NK cells was virtually confined to the spleen and liver. Binding of A-NK cells to liver neoplastic tissues was observed only after i.a. injections. CONCLUSION: This unique study design demonstrates that A-NK cells adoptively transferred to the liver via the intraarterial route have preferential access and substantial accumulation to the tumor site

Capitolo 4. I genitori delle ragazze e dei ragazzi LGB condividono, si interrogano e studiano

Che cosa vuol dire coming out? Come reagiscono i genitori al coming out? In che modo i genitori possono contribuire alla promozione del benessere della propria figlia o del proprio figlio? Sono alcune delle domande alle quali risponde questo libro attraverso le testimonianze di genitori di ragazze lesbiche e ragazzi gay. Ciò che emerge dalla lettura delle storie di vita è l’importanza di poter condividere le proprie emozioni ed esperienze. Il confronto con altri genitori in uno spazio di ascolto non giudicante permette non solo di superare le proprie paure e difficoltà ma soprattutto di rinascere come genitori

Inhibition of Leishmania infantum trypanothione reductase by NADPH, its natural substrate

Trypanothione reductase is a flavoprotein that catalyzes the β-NADPH-linked reduction of trypanothione (N1,N8-bis (glutathionyl)spermidine), a glutathione analog unique to trypanosomatid parasites [1]. The atomic structure of this enzyme from Leishmania infantum has been solved [2]. The enzyme contains a FAD moiety, a pair of redox-active cysteine residues (52 & 57) involved in trypanothione reduction, and two distinct binding sites for NADPH and trypanothione. Single- or double-mixing stopped-flow experiments in which trypanothione reductase is pre-mixed with either trypanothione or NADPH, indicate that turnover efficiency is critically dependent on the order of substrate addition. When trypanothione is allowed to occupy its binding site first, the enzyme obeys standard Michaelis-Menten kinetics with steady-state parameters kCAT = 68.0 0.2 s1 and KM for NADPH = 1.5 0.01 M. On the contrary, if the enzyme is pre-mixed with NADPH, which determines the formation of a characteristic charge-transfer complex between the FAD moiety and the proximal active-site sulfhydryl of Cys57, the enzyme enters a catalytically incompetent state whose lifetime depends on several factors. Preliminary Maldi-Tof mass spectrometry experiments performed on this NADPH-inactivated enzyme indicate that the mass of trypanothione reductase is increased by an amount consistent with the weight of trypanothione, an indication that trypanothione may have crosslinked to the polypeptide chain, at a position which is currently under investigation. Alltogether these results suggest that trypanothione reductase may undergo an auto-modification reaction during turnover allowing for the allosteric regulation of its enzymatic activity