Davide Corpillo

Matrix-Assisted Laser Desorption Ionization Imaging Mass Spectrometry Detection of a Magnetic Resonance Imaging Contrast Agent in Mouse Liver

The present paper describes the detection of a magnetic resonance imaging (MRI) contrast agent by matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS). The contrast agent was analyzed in both frozen and paraformaldehyde-fixed mouse livers explanted after its in vivo administration, and its identity was confirmed by fragmentation experiments. Moreover, a semiquantitative analysis was performed, evaluating its content in livers from mice sacrificed at different postadministration times. To the best of our knowledge, this is the first description of a MALDI-IMS analysis of MRI contrast agents and the first time that results obtained by MALDI-IMS are validated by both an in vivo (MRI) and an ex vivo (inductively coupled plasma atomic emission spectroscopy, ICP-AES) technique. Results shown in the present paper demonstrate the possibility of using MALDI-IMS for drug biodistribution analysis. Obviously, this application is particularly interesting in the case of unlabeled compounds, which cannot be detected by any of the other imaging techniques.

Lymphocyte proteomics of Parkinson’s disease patients reveals cytoskeletal protein dysregulation and oxidative stress

AIMS There is increasing evidence of biochemical alterations in peripheral blood lymphocytes of Parkinsons disease (PD) patients. In this work, we describe the changes in protein levels in peripheral lymphocytes of PD patients in order to identify potential peripheral biomarkers. MATERIALS & METHODS By means of 2D electrophoresis and mass spectrometry protein identification, we compared patients under L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, patients under subthalamic nucleus deep-brain stimulation and healthy controls. RESULTS Statistical analysis of the results demonstrated that cofilin-1, tropomyosin, and a specific actin isoform vary significantly in patients, regardless of the therapy. Two different isoforms of gamma-fibrinogen either correlate with the disease state or with the disease duration. Eventually, specific changes associated with the different therapies allowed to highlight oxidative stress conditions in lymphocytes in patients treated with higher doses of L-DOPA. CONCLUSIONS As a whole, peripheral blood lymphocytes are sensitive reporters of PD over inter-individual variability, and allow the identification of specific alterations that could be further exploited for diagnostic purposes.

Human SOD1-G93A specific distribution evidenced in murine brain of a transgenic model for amyotrophic lateral sclerosis by MALDI imaging mass spectrometry

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease caused by the degeneration of motor neurons. The transgenic mouse model carrying the human SOD1G93A mutant gene (hSOD1G93A mouse) represents one of the most reliable and widely used model of this pathology. In the present work, the innovative technique of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was applied in the study of pathological alterations at the level of small brain regions such as facial and trigeminal nuclei, which in rodents are extremely small and would be difficult to analyze with classical proteomics approaches. Comparing slices from three mice groups (transgenic hSOD1G93A, transgenic hSOD1WT, and nontransgenic, Ntg), this technique allowed us to evidence the accumulation of hSOD1G93A in the facial and trigeminal nuclei, where it generates aggregates. This phenomenon is likely to be correlated to the degeneration observed in these regions. Moreover, a statistical analysis allowed us to highlight other proteins as differentially expressed among the three mice groups analyzed. Some of them were identified by reverse-phase HPLC fractionation of extracted proteins and mass spectrometric analysis before and after trypsin digestion. In particular, the 40S ribosomal protein S19 (RPS19) was upregulated in the parenkyma and reactive glial cells in facial nuclei of hSOD1G93A mice when compared to transgenic hSOD1WT and nontransgenic ones.

Design and Synthesis of a γ1β8‐Cyclodextrin Oligomer: A New Platform with Potential Application as a Dendrimeric Multicarrier

We report the synthesis and characterization of a water-soluble, star-shaped macromolecular platform consisting of eight β-cyclodextrin (β-CD) units anchored to the narrower rim of a γ-CD core through bis(triazolyl)alkyl spacers. The efficient synthetic protocol is based on the microwave (MW)-promoted Cu-catalyzed 1,3-dipolar cycloaddition of CD monoazides to CD monoacetylenes. The ligand-hosting capability of the construct has been assessed by relaxometric titration and nuclear magnetic relaxation dispersion (NMRD) profiling, which showed it to be good, and this was supported by molecular dynamics simulations. To demonstrate the feasibility of obtaining supramolecular structures with high hosting ability, we designed a dimeric platform, formed by joining two nonamers through the γ-CD cores through a bis(lithocholic acid) linker. With a view to the potential biological applications, cytotoxicity and extent of binding to human serum albumin were assessed. The properties of this dendrimeric multicarrier make it suitable for pharmaceutical and diagnostic purposes, ranging from targeted drug delivery to molecular imaging.

The activity of aminoacyl-tRNA synthetase-interacting multi-functional protein 1 (AIMP1) on endothelial cells is mediated by the assembly of a cytoskeletal protein complex.

AIMP1 was first found as a factor associated with the aminoacyl‐tRNA synthetase (ARS) complex. However, it is also secreted and acts on different target cells such as endothelial cells, macrophages, and fibroblasts as an extracellular regulator, respectively, of angiogenesis, inflammatory responses and dermal regeneration. AIMP1 has also been reported to suppress in vivo tumor growth. In this study, we investigated the signaling pathways activated by exogenous AIMP1 in an in vitro endothelial model. AIMP1 decreases EC viability through an α5β1 integrin‐dependent mechanism and inhibits cell adhesion, is internalized and shows an asymmetric pattern of distribution and accumulation in cell protrusions. Experiments of affinity purification, pull down, and co‐immunoprecipitation showed that AIMP1 interacts with four cytoskeletal proteins (filamin‐A, α‐tubulin, vinculin, and cingulin). α‐Tubulin also gets phosphorylated upon cell treatment with AIMP1 and colocalization between AIMP1 and filamin‐A as well as between AIMP1 and cingulin was observed through immunofluorescence assays. In this work, we propose that AIMP1 effect on EC adhesion is mediated by the assembly of a cytoskeletal protein complex on the cytosolic face of the cell membrane which could regulate cellular architecture maintenance and remodeling. Moreover, this activity is able to indirectly influence cell viability. J. Cell. Biochem. 112: 1857–1868, 2011.

1H-NMR and MALDI-TOF MS as metabolomic quality control tests to classify platelet derived medium additives for GMP compliant cell expansion procedures

Introduction Ex vivo cell expansion under Good Manufacturing Practice (GMP) guidelines can be performed using medium additives containing human growth factors from platelets. These products can differently affect proliferation of adipose mesenchymal stromal stem cells (ASC). Qualification of medium additive performance is required for validation under GMP regulations: assessment of growth factor concentrations is not sufficient to predict the biological activity of the product batch. Proton nuclear magnetic resonance spectrometry (1H-NMR) and matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS) provide wide molecular characterization of samples. Aims We aimed to assess if 1H-NMR and MALDI-TOF MS techniques can be used as quality control test potentially predicting the impact of a medium additive on cell proliferation. Methods We tested the impact on ASC growth rate (cell proliferation assessment and cell morphology analysis) of four medium additives, obtained by different methods from human platelet apheresis product. In order to classify each medium additive, we evaluated growth factor concentrations and spectra obtained by 1H-NMR and by MALDI-TOF MS. Results Medium additive obtained by CaCl2 activation of platelet rich products induced higher proliferation rate vs additive derived from platelet depleted ones. Additives obtained by freeze-and-thaw methods weakly induced ASC proliferation. As expected, principal component analysis of growth factor concentrations did not unravel specific biochemical features characterizing medium additives in relation with their biological activity. Otherwise, while 1H-NMR showed a partial resolution capacity, analysis of MALDI-TOF MS spectra allowed unambiguous distinction between the medium additives we used to differently stimulate cell growth in vitro. Discussion MALDI-TOF and, despite limitations, 1H-NMR are promising cost effective and reliable quality controls to classify the potential of a medium additive to promote ASC growth. This can represent, after further investigations and appropriate validation, a significant advantage for GMP compliant manufacturing of advanced cell therapy products.

Corrigendum to “Validation of a mass spectrometry-based method for milk traces detection in baked food” [Food Chem. 199 (2016) 119–127]

The authors regret reversal of the order of given names and surnames of all authors which are now corrected as mentioned above. As a consequence the list of email addresses and author names is modified as shown below. The authors would like to apologise for any inconvenience caused