Dario Livio Longo

Iopamidol as a responsive MRI-chemical exchange saturation transfer contrast agent for pH mapping of kidneys: In vivo studies in mice at 7 T.

Iopamidol (Isovue®—Bracco Diagnostic Inc.) is a clinically approved X‐Ray contrast agent used in the last 30 years for a wide variety of diagnostic applications with a very good clinical acceptance. Iopamidol contains two types of amide functionalities that can be exploited for the generation of chemical exchange saturation transfer effect. The exchange rate of the two amide proton pools is markedly pH‐dependent. Thus, a ratiometric method for pH assessment has been set‐up based on the comparison of the saturation transfer effects induced by selective irradiation of the two resonances. This ratiometric approach allows to rule out the concentration effect of the contrast agent and provides accurate pH measurements in the 5.5–7.4 range. Upon injection of Iopamidol into healthy mice, it has been possible to acquire pH maps of kidney regions. Furthermore, it has been also shown that the proposed method is able to report about pH‐changes induced in control mice fed with acidified or basified water for a period of a week before image acquisition. Magn Reson Med, 2010.

Imaging the pH evolution of an acute kidney injury model by means of iopamidol, a MRI‐CEST pH‐responsive contrast agent

To investigate in vivo possible pH level alterations following an acute renal failure disease using a MRI‐CEST pH responsive contrast agent. The impact of functional evolution in different renal compartments over time was also investigated.

A general MRI-CEST ratiometric approach for pH imaging: demonstration of in vivo pH mapping with iobitridol.

Chemical exchange saturation transfer (CEST) is a novel contrast mechanism for magnetic resonance imaging (MRI). CEST MRI selectively saturates exchangeable protons that are transferred to MRI-detectable bulk water signal. MRI-CEST (pH)-responsive agents are probes able to map pH in the microenvironment in which they distribute. To minimize the confounding effects of contrast agent concentration, researchers have developed ratiometric CEST imaging, which investigates contrast agents containing multiple magnetically non-equivalent proton groups, whose prototropic exchange have different pH responses. However, conventional ratiometric CEST MRI imposes stringent requirements on the selection of CEST contrasts agents. In this study, a novel ratiometric pH MRI method based on the analysis of CEST effects under different radio frequency irradiation power levels was developed. The proposed method has been demonstrated using iobitridol, an X-ray contrast agent analog of iopamidol but containing a single set of amide protons, both in vitro and in vivo.

Methods for an improved detection of the MRI-CEST effect

CEST imaging is a recently introduced MRI contrast modality based on the use of endogenous or exogenous molecules whose exchangeable proton pools transfer saturated magnetization to bulk water, thus creating negative contrast. One of the critical issues for further development of these agents is represented by their limited sensitivity in vivo. The aim of this work is to improve the detection of CEST agents by exploring new approaches through which the saturation transfer (ST) effect can be enhanced. The performance of the proposed methods has been tested in vitro and in vivo using highly sensitive and highly shifted lipoCEST agents, and the results were compared with the standard ST evaluation mode. The acquired Z-spectra were interpolated locally and voxel-by-voxel by smoothing splines. Besides expressing the ST in the standard mode, we explore two methods, enhanced and integral ST, which better exploit all the information contained in the Z-spectrum. By combining different modes for ST assessment a significant improvement in the detection of the lipoCEST agents, both in vitro and in vivo, has been found. The results obtained from the application of the proposed methods outline the importance of post-processing analysis for highlighting the CEST-MRI contrast.

Advances in Metal-Based Probes for MR Molecular Imaging Applications

The role of MRI in the armory of diagnostic modalities for the medicine of the forthcoming years largely depends on how chemistry will provide advanced tools to meet the medical needs. This review aims at outlining the most innovative approaches that have been undertaken in the recent history of MRI contrast agents for tackling the challenges of sensitivity and specificity required by the new generation of contrast agents that should allow the visualization of pathological processes occurring on cellular and molecular scale (the so-called Molecular Imaging). Most of the classes of MRI agents clinically approved or currently under investigation in a preclinical phase exploit peculiar magnetic properties of metals. The conventional agents acting as T(1) or T(2)/T(2)* relaxation enhancers are primarily based on the paramagnetic or the superparamagnetic properties of Gd(III)-, Mn(II)- and iron oxides systems. Recently, there has been a renewed interest towards paramagnetic lanthanide complexes with an anisotropic electronic configuration thanks to their ability to induce strong effect on the resonance frequency of the spins dipolarly coupled with them. Such systems, formerly mainly used as shift reagents, have now attracted much attention in the emerging field of Chemical Exchange Saturation Transfer (CEST) MRI agents.

PI3K-C2γ is a Rab5 effector selectively controlling endosomal Akt2 activation downstream of insulin signalling

In the liver, insulin-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is at the core of metabolic control. Multiple PI3K and Akt isoenzymes are found in hepatocytes and whether isoform-selective interplays exist is currently unclear. Here we report that insulin signalling triggers the association of the liver-specific class II PI3K isoform γ (PI3K-C2γ) with Rab5-GTP, and its recruitment to Rab5-positive early endosomes. In these vesicles, PI3K-C2γ produces a phosphatidylinositol-3,4-bisphosphate pool specifically required for delayed and sustained endosomal Akt2 stimulation. Accordingly, loss of PI3K-C2γ does not affect insulin-dependent Akt1 activation as well as S6K and FoxO1-3 phosphorylation, but selectively reduces Akt2 activation, which specifically inhibits glycogen synthase activity. As a consequence, PI3K-C2γ-deficient mice display severely reduced liver accumulation of glycogen and develop hyperlipidemia, adiposity as well as insulin resistance with age or after consumption of a high-fat diet. Our data indicate PI3K-C2γ supports an isoenzyme-specific forking of insulin-mediated signal transduction to an endosomal pool of Akt2, required for glucose homeostasis.

Nanoparticle-based chemical exchange saturation transfer (CEST) agents.

The frequency‐encoding property of chemical exchange saturation transfer (CEST) agents places them in a unique position among the MRI contrast agents, as it allows the visualization of more agents in the same MR image, as well as making it possible to set up innovative MRI‐responsive agents. The sensitivity issue shown by molecular CEST agents (either diamagnetic or paramagnetic) has been tackled with the design of nanoparticle‐based CEST agents endowed with a large number of exchangeable protons that ensure large saturation transfer levels. Several systems have been considered, namely supramolecular adducts, dendrimers, micelles and liposomes loaded with CEST agents (in most cases, paramagnetic CEST agents). A particularly sensitive system is represented by lipoCEST agents, consisting of liposomes whose inner water resonance is shifted by the co‐presence of paramagnetic lanthanide complexes. The exchangeable pool of protons is represented by all the water molecules contained in the inner liposome cavity (106–108). Although in vitro work has provided excellent results, in vivo translation appears to be hampered to some extent by the peculiar behavior shown by these particles on administration to living animals. Copyright

Structural and functional differences between KRIT1A and KRIT1B isoforms: a framework for understanding CCM pathogenesis

KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed. Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein-protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms. We found that the 15th exon encodes for the distal beta-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner. As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure-function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.

Magnetic resonance imaging visualization of targeted cells by the internalization of supramolecular adducts formed between avidin and biotinylated Gd3+ chelates

The high binding affinity between avidin and biotin has been exploited to develop a procedure for magnetic resonance imaging (MRI) visualization of target cells. SHIN3 and PANC1 tumor cell lines have been used as target cells because they possess on their membranes galactosyl receptors able to bind avidin molecules. Avidin–Gd chelate adducts have been built by using two Gd complexes containing one (Gd-I) and two (Gd-II) biotin residues, respectively. The relaxivities of such supramolecular adducts are significantly higher than those shown by free Gd-I and Gd-II. There is evidence of the occurrence of multilayered adducts in which the bis-biotinylated Gd3+ complex acts as a bridge between adjacent avidin molecules. MRI differentiation of labeled versus unlabeled cells has been attained when approximately 6×108 Gd units were internalized in each cell. Furthermore, there is a marked decrease in the measured intracellular T1 relaxivity as the number of internalized Gd complexes increases, probably owing to too short relaxation times of endosomic water protons with respect to their diffusion lifetime.

New Insights for Pursuing High Relaxivity MRI Agents from Modelling the Binding Interaction of GdIII Chelates to HSA

It was recognized early on that the relaxivity of a Gd complex at 0.5–1 T can be strongly enhanced if its molecular correlation time is lengthened by linking it to a slowly moving macromolecule. 2] In this context, a huge amount of attention has been devoted in the past decade to the study of systems able to form noncovalent adducts with serum albumin, which also has the advantage of yielding systems that remain confined in the blood vessels. Theory foresees the attainment of relaxivities >100 mm 1 s 1 for macromolecular monoaquo Gd chelates characterized by a molecular reorientation time of 10–30 ns. 6] However, in spite of a number of investigated systems, relaxivities of such magnitude for Gd chelates bound to HSA have never been found. One major limiting factor has been recognized to be the occurrence of an insufficiently fast exchange rate of the coordinated water (tM). [7, 8]