Publications by authors named "Massimo Castagnola"

175 Publications

Oxidative and Proteolytic Inactivation of Alpha-1 Antitrypsin in Bronchopulmonary Dysplasia Pathogenesis: A Top-Down Proteomic Bronchoalveolar Lavage Fluid Analysis.

Front Pediatr 2021 23;9:597415. Epub 2021 Mar 23.

Dipartimento di Scienze della Vita e Sanità Pubblica, Unità Operativa Complessa di Neonatologia, Fondazione Policlinico Universitario A. Gemelli, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy.

The study investigates the role of the oxidative and proteolytic inactivation of alpha-1 antitrypsin (AAT) in the pathogenesis of bronchopulmonary dysplasia (BPD) in premature infants. Bronchoalveolar lavage fluid (BALF) samples were collected on the 3rd day of life from mechanically ventilated neonates with gestational age ≤ 30 weeks and analyzed without previous treatment (top-down proteomics) by reverse-phase high-performance liquid chromatography-electrospray ionization mass spectrometry. AAT fragments were identified by high-resolution LTQ Orbitrap XL experiments and the relative abundances determined by considering the extracted ion current (XIC) peak area. Forty preterm neonates were studied: 20 (50%) did not develop BPD (no-BPD group), 17 (42.5%) developed mild or moderate new-BPD (mild + moderate BPD group), and 3 (7.5%) developed severe new-BPD (severe BPD group). Eighteen fragments of AAT and a fragment of AAT oxidized at a methionine residue were identified: significantly higher values of AAT fragments 25-57, 375-418, 397-418, 144-171, and 397-418 with oxidized methionine were found in the severe BPD group. The significantly higher levels of several AAT fragments and of the fragment 397-418, oxidized in BALF of preterm infants developing BPD, underlie the central role of an imbalance between proteases and protease inhibitors in exacerbating lung injury and inducing most severe forms of BPD. The study has some limitations, and between them, the small sample size implies the need for further confirmation by larger studies.
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http://dx.doi.org/10.3389/fped.2021.597415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8021761PMC
March 2021

90Y-DOTA-nimotuzumab: Synthesis of a Promising β⁻ radiopharmaceutical.

Curr Radiopharm 2021 Jan 4. Epub 2021 Jan 4.

Unit of Nuclear Medicine, Fondazione Policlinico Universitario A. Gemelli IRCCS, Roma. Italy.

Background: Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) monoclonal antibody, nowadays used for tumour immunochemotherapy. This study aimed to label the conjugate DOTA-nimotuzumab with yttrium-90, in order to provide a β- emitting radioimmunoconjugate (90Y-DOTA-nimotuzumab) potentially useful to assess the feasibility of a new radio-guided surgery approach.

Methods: The synthesis of 90Y-DOTA-nimotuzumab was performed in two days. Nimotuzumab was conjugated with a 50 fold excess of DOTA and then labelled with 90Y3+. The 90Y-DOTA-nimotuzumab preparation was optimized considering several parameters such as pH, temperature and reaction volume. Moreover, the 90Y-DOTA-nimotuzumab stability was evaluated in human plasma.

Results: The radioimmunoconjugate 90Y-DOTA-nimotuzumab was obtained with a radiochemical purity greater than 96%, and showed a good stability at 20°C as well as at 37°C in human plasma.

Conclusions: The optimized conditions for a mild and easy preparation of 90Y-DOTA-nimotuzumab joined to a promising stability under physiological conditions suggest to propose this radioimmunoconjugate as a potential diagnostic radiopharmaceutical for β- radio-guided surgery.
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http://dx.doi.org/10.2174/1874471013999210104220031DOI Listing
January 2021

HPLC-ESI-MS top-down analysis of salivary peptides of preterm newborns evidenced high activity of some exopeptidases and convertases during late fetal development.

Talanta 2021 Jan 25;222:121429. Epub 2020 Aug 25.

Laboratorio di Proteomica, Centro Europeo di Ricerca Sul Cervello, IRCCS Fondazione Santa Lucia, Roma, Italy. Electronic address:

To have information on the proteolytic activity of convertases and exo-peptidases on human salivary proteins, this study investigated the relative amounts of the truncated proteoforms in the saliva of preterm newborns and compared them with the relative amounts measured in saliva of at-term newborns, of babies (0-10 years old) and of adults. Results indicated that convertase(s), acting on acidic proline-rich proteins and histatin 3, and carboxypeptidase(s) acting on acidic proline-rich proteins, P-C peptide, histatin 6 and statherin were many folds more active in preterm newborns than in the other groups. Conversely, the aminopeptidase responsible for the removal of the N-terminal Asp residue of statherin was not active in preterm newborns, becoming active only several months after the normal term of delivery. The high activity of convertases determined in preterm newborns suggests that it is required for the molecular events connected to the fetus development, and encourages further studies devoted to the characterization of their specific substrates.
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http://dx.doi.org/10.1016/j.talanta.2020.121429DOI Listing
January 2021

Top down proteomic analysis of gingival crevicular fluid in deciduous, exfoliating and permanent teeth in children.

J Proteomics 2020 08 3;226:103890. Epub 2020 Jul 3.

Laboratorio di Proteomica e Metabonomica-IRCCS Fondazione Santa Lucia, Roma, Italy.

Gingival Crevicular Fluid (GCF), a plasma-derived exudate present in the gingival crevice was collected from deciduous, exfoliating and permanent teeth from 20 children (60 samples) with the aim to characterize and quantify by a mass spectrometry based top-down proteomic approach, the peptide/proteins in the fluid and verify possible variations occurring during the exfoliating process. The results obtained confirmed the presence in GCF of α-Defensins 1-4, Thymosin β4 and Thymosin β10, as described in previous works and revealed the presence of other interesting peptides never described before in GCF such as specific fragments of α-1-antitrypsin, α-1-antichymotrypsin; fragments of Thymosin β4 and Thymosin β10; Fibrinopeptide A and its fragments and Fibrinopeptide B; S100A8 and S100A9, LVV Hemorphin-7 (hemoglobin chain β fragment), as well as some other peptides deriving from α and β subunits of hemoglobin. Statistical analysis evidenced different levels in 5 proteins/peptides in the three groups. Our study demonstrate that an in-depth analysis of a biological fluid like GCF, present in small amount, can provide useful information for the understanding of different biological processes like teeth eruption. Data are available via ProteomeXchange with identifier PXD016010 and PXD016049. SIGNIFICANCE: GCF due to his site-specific nature has a great potential in containing factors that are specific for action at a given site and might have diagnostic value to detect qualitative and quantitative variations of proteins/peptides composition linked to physiological or pathological conditions.
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http://dx.doi.org/10.1016/j.jprot.2020.103890DOI Listing
August 2020

Adamantinomatous craniopharyngioma: advances in proteomic research.

Childs Nerv Syst 2021 Mar 2;37(3):789-797. Epub 2020 Jul 2.

UOC Neurochirurgia Infantile, Dipartimento di Scienze dell'Invecchiamento, Neurologiche, Ortopediche e della Testa-Collo; Fondazione Policlinico Universitario A. Gemelli - IRCCS, Università Cattolica del Sacro Cuore, Largo Gemelli 1, 00168, Rome, Italy.

Background: Many efforts have been performed in the last decade to accomplish the genomic and proteomic characterization of pediatric adamantinomatous craniopharyngioma with the purpose to elucidate the molecular mechanisms underlying the onset and development of this pediatric brain tumor, its high recurrence rate, and, although classified as a histologically benign neoplasm, its aggressive behavior.

Methods: The focus of this review is to perform the new comparison of the proteomic profiles of the solid component and the intracystic fluid of adamantinomatous craniopharyngioma based on our previous results, obtained by both the top-down and the bottom-up proteomic approaches, to disclose differences and similarities, and to discuss the results in the context of the most recent literature.

Results And Conclusions: Proteins and peptides identified in the cyst fluid and in the solid component of adamantinomatous craniopharyngioma (AC) include beyond markers of inflammation (i.e., alpha-defensins), proteins involved in cell migration and protein degradation (i.e., beta-thymosin and ubiquitin peptides), whose main role might be in tumor growth and infiltration of the surrounding neural structures. These last appeared different in the solid components compared with the cyst fluid, missing their terminal part in the solid tissue, a feature generally associated to malignancies, which might represent a distinct molecular site for an aggressive behavior of AC.
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http://dx.doi.org/10.1007/s00381-020-04750-zDOI Listing
March 2021

Multiple Herpes Simplex Virus-1 (HSV-1) Reactivations Induce Protein Oxidative Damage in Mouse Brain: Novel Mechanisms for Alzheimer's Disease Progression.

Microorganisms 2020 Jun 29;8(7). Epub 2020 Jun 29.

Department of Public Health and Infectious Diseases, Sapienza University of Rome, Laboratory affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, 00185 Rome, Italy.

Compelling evidence supports the role of oxidative stress in Alzheimer's disease (AD) pathophysiology. Interestingly, Herpes simplex virus-1 (HSV-1), a neurotropic virus that establishes a lifelong latent infection in the trigeminal ganglion followed by periodic reactivations, has been reportedly linked both to AD and to oxidative stress conditions. Herein, we analyzed, through biochemical and redox proteomic approaches, the mouse model of recurrent HSV-1 infection we previously set up, to investigate whether multiple virus reactivations induced oxidative stress in the mouse brain and affected protein function and related intracellular pathways. Following multiple HSV-1 reactivations, we found in mouse brains increased levels of oxidative stress hallmarks, including 4-hydroxynonenal (HNE), and 13 HNE-modified proteins whose levels were found significantly altered in the cortex of HSV-1-infected mice compared to controls. We focused on two proteins previously linked to AD pathogenesis, i.e., glucose-regulated protein 78 (GRP78) and collapsin response-mediated protein 2 (CRMP2), which are involved in the unfolded protein response (UPR) and in microtubule stabilization, respectively. We found that recurrent HSV-1 infection disables GRP78 function and activates the UPR, whereas it prevents CRMP2 function in mouse brains. Overall, these data suggest that repeated HSV-1 reactivation into the brain may contribute to neurodegeneration also through oxidative damage.
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http://dx.doi.org/10.3390/microorganisms8070972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409037PMC
June 2020

Top-Down Proteomics of Human Saliva Discloses Significant Variations of the Protein Profile in Patients with Mastocytosis.

J Proteome Res 2020 08 6;19(8):3238-3253. Epub 2020 Jul 6.

Dipartimento di Scienze della Vita e dell'Ambiente, Università di Cagliari, 09124 Cagliari, Italy.

Mastocytosis is a myeloproliferative neoplasm causing abnormal clonal mast cell accumulation in different tissues, such as skin and bone marrow. A cutaneous subtype (CM) is distinguished from a systemic one (SM); SM patients can be grouped into SM with (SM+C) or without (SM-C) additional cutaneous lesions, and their classification is often challenging. This study was purposed to highlight variations in the salivary proteome of patients with different mastocytosis subtypes and compared to healthy controls. A top-down proteomics approach coupled to a label-free quantitation revealed salivary profiles in patients different from those of controls and a down-regulation of peptides/proteins involved in the mouth homeostasis and defense, such as statherin, histatins, and acidic proline-rich proteins (aPRPs), and in innate immunity and inflammation, such as the cathepsin inhibitors, suggesting a systemic condition associated with an exacerbated inflammatory state. The up-regulation of antileukoproteinase and S100A8 suggested a protective role against the disease status. The two SM forms were distinguished by the lower levels of truncated forms of aPRPs, statherin, P-B peptide, and cystatin D and the higher levels of thymosin β4 and α-defensins 1 and 4 in SM-C patients with respect to SM+C. Data are available via ProteomeXchange with identifier PXD017759.
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http://dx.doi.org/10.1021/acs.jproteome.0c00207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008451PMC
August 2020

Proteomic Analysis of the Acid-Insoluble Fraction of Whole Saliva from Patients Affected by Different Forms of Non-histaminergic Angioedema.

J Clin Immunol 2020 08 10;40(6):840-850. Epub 2020 Jun 10.

Dept of Life and Environmental Sciences, University of Cagliari, 09042, Monserrato, CA, Italy.

We analyzed by bidimensional electrophoresis the acid-insoluble fraction of saliva from three classes of angioedema patients and a healthy control group, highlighting significant variations of several normalized spot volumes. Characterization of the corresponding proteins was performed by in-gel tryptic digestion of the spots, followed by high-resolution HPLC-ESI-MS/MS analysis of tryptic mixtures. By this strategy, 16 differentially-expressed proteins among two or more groups were identified. We found higher concentration of proteins involved in immune response (interleukin-1 receptor antagonist and annexin A1), and of moonlighting proteins acting as plasminogen receptors (glyceraldehyde-3-phosphate dehydrogenase, α-enolase, and annexin A2) in patients affected by the idiopathic non-histaminergic or hereditary angioedema with unknown origin with respect to healthy controls. These data provide new information on the molecular basis of these less characterized types of angioedema. Graphical Abstract Graphical Abstract.
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http://dx.doi.org/10.1007/s10875-020-00802-wDOI Listing
August 2020

Ultra-rapid glutathionylation of chymotrypsinogen in its molten globule-like conformation: A comparison to archaeal proteins.

Sci Rep 2020 06 2;10(1):8943. Epub 2020 Jun 2.

Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma "Tor Vergata", Rome, Italy.

Chymotrypsinogen, when reduced and taken to its molten globule-like conformation, displays a single cysteine with an unusual kinetic propensity toward oxidized glutathione (GSSG) and other organic thiol reagents. A single residue, identified by mass spectrometry like Cys1, reacts with GSSG about 1400 times faster than an unperturbed protein cysteine. A reversible protein-GSSG complex and a low pK (8.1 ± 0.1) make possible such astonishing kinetic property which is absent toward other natural disulfides like cystine, homocystine and cystamine. An evident hyper-reactivity toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 1-chloro-2,4-dinitrobenzene (CDNB) was also found for this specific residue. The extraordinary reactivity toward GSSG is absent in two proteins of the thermophilic archaeon Sulfolobus solfataricus, an organism lacking glutathione: the Protein Disulphide Oxidoreductase (SsPDO) and the Bacterioferritin Comigratory Protein 1 (Bcp1) that displays Cys residues with an even lower pK value (7.5 ± 0.1) compared to chymotrypsinogen. This study, which also uses single mutants in Cys residues for Bcp1, proposes that this hyper-reactivity of a single cysteine, similar to that found in serum albumin, lysozyme, ribonuclease, may have relevance to drive the "incipit" of the oxidative folding of proteins from organisms where the glutathione/oxidized glutathione (GSH/GSSG) system is present.
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http://dx.doi.org/10.1038/s41598-020-65696-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265447PMC
June 2020

Exploring the HeLa Dark Mitochondrial Proteome.

Front Cell Dev Biol 2020 5;8:137. Epub 2020 Mar 5.

Proteomics and Metabonomics Unit, IRCCS-Fondazione Santa Lucia, Rome, Italy.

In the framework of the Human Proteome Project initiative, we aim to improve mapping and characterization of mitochondrial proteome. In this work we implemented an experimental workflow, combining classical biochemical enrichments and mass spectrometry, to pursue a much deeper definition of mitochondrial proteome and possibly mine mitochondrial uncharacterized . We fractionated in two compartments mitochondria enriched from HeLa cells in order to annotate 4230 proteins in both fraction by means of a multiple-enzyme digestion (trypsin, chymotrypsin and Glu-C) followed by mass spectrometry analysis using a combination of Data Dependent Acquisition (DDA) and Data Independent Acquisition (DIA). We detected 22 mitochondrial dark proteins not annotated for their function and we provide their relative abundance inside the mitochondrial organelle. Considering this work as a pilot study we expect that the same approach, in different biological system, could represent an advancement in the characterization of the human mitochondrial proteome providing uncharted ground to explore the mitonuclear phenotypic relationships. All spectra have been deposited to ProteomeXchange with PXD014201 and PXD014200 identifier.
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http://dx.doi.org/10.3389/fcell.2020.00137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066081PMC
March 2020

Altered mitochondrial function in cells carrying a premutation or unmethylated full mutation of the FMR1 gene.

Hum Genet 2020 Feb 9;139(2):227-245. Epub 2020 Jan 9.

Istituto di Medicina Genomica, Università Cattolica del Sacro Cuore, Fondazione Policlinico Universitario A. Gemelli IRCCS, Largo F. Vito 1, 00168, Roma, Italy.

Fragile X-related disorders are due to a dynamic mutation of the CGG repeat at the 5' UTR of the FMR1 gene, coding for the RNA-binding protein FMRP. As the CGG sequence expands from premutation (PM, 56-200 CGGs) to full mutation (> 200 CGGs), FMRP synthesis decreases until it is practically abolished in fragile X syndrome (FXS) patients, mainly due to FMR1 methylation. Cells from rare individuals with no intellectual disability and carriers of an unmethylated full mutation (UFM) produce slightly elevated levels of FMR1-mRNA and relatively low levels of FMRP, like in PM carriers. With the aim of clarifying how UFM cells differ from CTRL and FXS cells, a comparative proteomic approach was undertaken, from which emerged an overexpression of SOD2 in UFM cells, also confirmed in PM but not in FXS. The SOD2-mRNA bound to FMRP in UFM more than in the other cell types. The high SOD2 levels in UFM and PM cells correlated with lower levels of superoxide and reactive oxygen species (ROS), and with morphological anomalies and depolarization of the mitochondrial membrane detected through confocal microscopy. The same effect was observed in CTRL and FXS after treatment with MC2791, causing SOD2 overexpression. These mitochondrial phenotypes reverted after knock-down with siRNA against SOD2-mRNA and FMR1-mRNA in UFM and PM. Overall, these data suggest that in PM and UFM carriers, which have high levels of FMR1 transcription and may develop FXTAS, SOD2 overexpression helps to maintain low levels of both superoxide and ROS with signs of mitochondrial degradation.
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http://dx.doi.org/10.1007/s00439-019-02104-7DOI Listing
February 2020

RP-HPLC-ESI-IT Mass Spectrometry Reveals Significant Variations of the Human Salivary Protein Profile Associated with Predominantly Antibody Deficiencies.

J Clin Immunol 2020 02 8;40(2):329-339. Epub 2020 Jan 8.

Department of Life and Environmental Sciences, University of Cagliari, Cittadella Univ. Monserrato, ss 554, 09042, Monserrato, CA, Italy.

Purpose: Present study is designed to discover potential salivary biomarkers associated with predominantly antibody deficiencies, which include a large spectrum of disorders sharing failure of antibody production, and B cell defects resulting in recurrent infections, autoimmune and inflammatory manifestations, and tumor susceptibility. Understanding and clinical classification of these syndromes is still challenging.

Methods: We carried out a study of human saliva based on liquid chromatography-mass spectrometry measurements of intact protein mass values. Salivary protein profiles of patients (n = 23) and healthy controls (n = 30) were compared.

Results: Patients exhibited lower abundance of α-defensins 1-4, cystatins S1 and S2, and higher abundance of glutathionylated cystatin B and cystatin SN than controls. Patients could be clustered in two groups on the basis of different levels of cystatin SN, S1 and S2, suggesting that these proteins may play different roles in the disease.

Conclusions: Quantitative variations of these pro-inflammatory and antimicrobial peptides/proteins may be related to immunodeficiency and infectious condition of the patients. The high incidence of tumors in the group with the highest level of cystatin SN, which is recognized as tumoral marker, appeared an intriguing result deserving of future investigations. Data are available via ProteomeXchange with identifier PXD012688.
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http://dx.doi.org/10.1007/s10875-020-00743-4DOI Listing
February 2020

Protein Oxidative Damage in UV-Related Skin Cancer and Dysplastic Lesions Contributes to Neoplastic Promotion and Progression.

Cancers (Basel) 2020 01 1;12(1). Epub 2020 Jan 1.

Department of RiDAIT, the Regina Elena National Cancer Institute IRCCS, via Elio Chianesi 53, 00144 Rome, Italy.

The ultraviolet (UV) component of solar radiation is the major driving force of skin carcinogenesis. Most of studies on UV carcinogenesis actually focus on DNA damage while their proteome-damaging ability and its contribution to skin carcinogenesis have remained largely underexplored. A redox proteomic analysis of oxidized proteins in solar-induced neoplastic skin lesion and perilesional areas has been conducted showing that the protein oxidative burden mostly concerns a selected number of proteins participating to a defined set of functions, namely: chaperoning and stress response; protein folding/refolding and protein quality control; proteasomal function; DNA damage repair; protein- and vesicle-trafficking; cell architecture, adhesion/extra-cellular matrix (ECM) interaction; proliferation/oncosuppression; apoptosis/survival, all of them ultimately concurring either to structural damage repair or to damage detoxication and stress response. In peri-neoplastic areas the oxidative alterations are conducive to the persistence of genetic alterations, dysfunctional apoptosis surveillance, and a disrupted extracellular environment, thus creating the condition for transformant clones to establish, expand and progress. A comparatively lower burden of oxidative damage is observed in neoplastic areas. Such a finding can reflect an adaptive selection of best fitting clones to the sharply pro-oxidant neoplastic environment. In this context the DNA damage response appears severely perturbed, thus sustaining an increased genomic instability and an accelerated rate of neoplastic evolution. In conclusion UV radiation, in addition to being a cancer-initiating agent, can act, through protein oxidation, as a cancer-promoting agent and as an inducer of genomic instability concurring with the neoplastic progression of established lesions.
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http://dx.doi.org/10.3390/cancers12010110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017152PMC
January 2020

Quantitative Analysis of the Seminal Plasma Proteome in Secondary Hypogonadism.

J Clin Med 2019 Dec 3;8(12). Epub 2019 Dec 3.

International Scientific Institute "Paul VI", 100168 Rome, Italy.

In the grey zone of testosterone levels between 8 and 12 nmol/L, the usefulness of therapy is controversial; as such, markers of tissue action of androgens may be helpful in adjusting clinical decisions. To better understand the effect of the hypothalamic-pituitary-testicular axis on male accessory secretion, we performed a proteomic quantitative analysis of seminal plasma in patients with secondary hypogonadism, before and after testosterone replacement therapy (TRT). Ten male patients with postsurgical hypogonadotrophic hypogonadism were enrolled in this study, and five of these patients were evaluated after testosterone treatment. Ten men with proven fertility were selected as a control group. An aliquot of seminal plasma from each individual was subjected to an in-solution digestion protocol and analyzed using an Ultimate 3000 RSLC-nano HPLC apparatus coupled to a LTQ Orbitrap Elite mass spectrometer. The label-free quantitative analysis was performed via Precursor Ions Area Detector Node. Eleven proteins were identified as decreased in hypogonadic patients versus controls, which are primarily included in hydrolase activity and protein binding activity. The comparison of the proteome before and after TRT comes about within the discovery of six increased proteins. This is the primary application of quantitative proteomics pointed to uncover a cluster of proteins reflecting an impairment not only of spermatogenesis but of the epididymal and prostate epithelial cell secretory function in male hypogonadism. The identified proteins might represent putative clinical markers valuable within the follow-up of patients with distinctive grades of male hypogonadism.
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http://dx.doi.org/10.3390/jcm8122128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947469PMC
December 2019

Mapping of Transglutaminase-2 Sites of Human Salivary Small Basic Proline-Rich Proteins by HPLC-High-Resolution ESI-MS/MS.

J Proteome Res 2020 01 6;19(1):300-313. Epub 2019 Nov 6.

Department of Life and Environmental Sciences , University of Cagliari, Cittadella Univ. Monserrato , Monserrato, Cagliari 09042 , Italy.

Because of the distinctive features of the oral cavity, the determination of the proteins involved in the formation of the "oral protein pellicle" is demanding. The present study investigated the susceptibility of several human basic proline-rich peptides, named P-H, P-D, P-F, P-J, and II-2, as substrates of transglutaminase-2. The reactivity of the P-C peptide and statherin was also investigated. Peptides purified from human whole saliva were incubated with the enzyme in the presence or in the absence of monodansyl-cadaverine. Mass spectrometry analyses of the reaction products highlighted that P-H and P-D (P and A variants) were active substrates, II-2 was less reactive, and P-F and P-J showed very low reactivity. P-C and statherin were highly reactive. All of the peptides formed cyclo derivatives, and only specific glutamine residues were involved in the cycle formation and reacted with monodansyl-cadaverine: Q of P-H, Q of P-D, Q of II-2, Q of P-C, and Q of statherin were the principal reactive residues. One or two secondary glutamine residues of only P-H, P-D P, P-C, and statherin were hierarchically susceptible to the reaction with monodansyl-cadaverine. MS and MS/MS data were deposited to the ProteomeXchange Consortium ( http://www.ebi.ac.uk/pride ) via the PRIDE partner repository with the data set identifier PXD014658.
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http://dx.doi.org/10.1021/acs.jproteome.9b00527DOI Listing
January 2020

Enrichments of post-translational modifications in proteomic studies.

J Sep Sci 2020 Jan 6;43(1):313-336. Epub 2019 Nov 6.

Dipartimento di Scienze della Vita e dell'Ambiente, Università di Cagliari, Cagliari, Italy.

More than 300 different protein post-translational modifications are currently known, but only a few have been extensively investigated because modified proteoforms are commonly present in sub-stoichiometry amount. For this reason, improvement of specific enrichment techniques is particularly useful for the proteomic characterization of post-translationally modified proteins. Enrichment proteomic strategies could help the researcher in the challenging issue to decipher the complex molecular cross-talk existing between the different factors influencing the cellular pathways. In this review the state of art of the platforms applied for the enrichment of specific and most common post-translational modifications, such as glycosylation and glycation, phosphorylation, sulfation, redox modifications (i.e. sulfydration and nitrosylation), methylation, acetylation, and ubiquitinylation, are described. Enrichments strategies applied to characterize less studied post-translational modifications are also briefly discussed.
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http://dx.doi.org/10.1002/jssc.201900804DOI Listing
January 2020

Exploring the brain tissue proteome of TgCRND8 Alzheimer's Disease model mice under B vitamin deficient diet induced hyperhomocysteinemia by LC-MS top-down platform.

J Chromatogr B Analyt Technol Biomed Life Sci 2019 Aug 5;1124:165-172. Epub 2019 Jun 5.

Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Rome, Italy. Electronic address:

The multifactorial nature of Late Onset Alzheimer's Disease (LOAD), the AD form of major relevance on epidemiological and social aspects, has driven the original investigation by LC-MS and top-down proteomics approach of the protein repertoire of the brain tissue of TgCRND8 model mice fed with a diet deficient in B vitamins. The analysis of the acid-soluble fraction of brain tissue homogenates identified a list of proteins and peptides, proteoforms and PTMs. In order to disclose possible modulations, their relative quantification in wild type and AD model mice under both B vitamin deficient and control diets was performed. The levels of metallothionein III, guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 and brain acid soluble protein 1 showed statistically significant alterations depending on genotype, diet or both effects, respectively. Particularly, metallothionein III exhibited increased levels in TgCRND8 mice under B vitamin deficient diet with respect to wild type mice under both diets. Brain acid soluble protein 1 showed the opposite, revealing decreased levels in all diet groups of AD model mice with respect to wild type mice in control diet. Lower levels of brain acid soluble protein 1 were also observed in wild type mice under deficiency of B vitamins. These results, besides contributing to increase the knowledge of AD at molecular level, give new suggestions for deeply investigating metallothionein III and brain acid soluble protein 1 in AD.
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http://dx.doi.org/10.1016/j.jchromb.2019.06.005DOI Listing
August 2019

Investigating the Protein Signature of Adamantinomatous Craniopharyngioma Pediatric Brain Tumor Tissue: Towards the Comprehension of Its Aggressive Behavior.

Dis Markers 2019 2;2019:3609789. Epub 2019 May 2.

Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Roma, Italy.

Although histologically benign, adamantinomatous craniopharyngioma (AC) pediatric brain tumor is a locally aggressive disease that frequently determines symptoms and hormonal dysfunctions related to the mass effect on the surrounding structures. Another typical feature of this benign neoplasm is the presence of voluminous liquid cysts frequently associated with the solid component. Even if studies have been devoted to the proteomic characterization of the tumor intracystic fluid, poor explorations have been performed on its solid part, principally investigated by transcriptomics technologies. In the present study, seven specimens of AC whole tumor tissue have been analyzed by LC-MS for a preliminary assessment of the proteomic profile by a top-down/bottom-up integrated approach. Thymosin beta 4, ubiquitin, calmodulin, S100 proteins, prothymosin isoform 2, alpha-defensins 1-4, and fragments largely belonging to vimentin, hemoglobin, and glial fibrillary acidic protein characterized the intact proteome. The identification of alpha-defensins, formerly characterized in AC intracystic fluid, reinforces the hypothesis of a role for inflammation in tumor pathogenesis. A total number of 1798 unique elements were identified by a bottom-up approach with a special focus on the 433 proteins commonly characterized in the 85.7% of the samples analyzed. Their gene ontology classification evidenced the involvement of the adherence system, intermediate filaments, and actin cytoskeleton in tumor pathogenesis and of elements part of the Wnt, FGF, and EGFR signaling pathways. In addition, proteins involved in calcium modulation, innate immunity, inflammation, CCKR and integrin signaling, and gonadotropin-releasing hormone receptor pathways were also outlined. Further than confirming proteomic data previously obtained on AC intracystic fluid, these results offer a preliminary overview of the AC whole tissue protein phenotype, adding new hints towards the comprehension of this still obscure pediatric brain tumor.
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http://dx.doi.org/10.1155/2019/3609789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525946PMC
December 2019

Restoration of aberrant mTOR signaling by intranasal rapamycin reduces oxidative damage: Focus on HNE-modified proteins in a mouse model of down syndrome.

Redox Biol 2019 05 9;23:101162. Epub 2019 Mar 9.

Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy. Electronic address:

Increasing evidences support the notion that the impairment of intracellular degradative machinery is responsible for the accumulation of oxidized/misfolded proteins that ultimately results in the deposition of protein aggregates. These events are key pathological aspects of "protein misfolding diseases", including Alzheimer disease (AD). Interestingly, Down syndrome (DS) neuropathology shares many features with AD, such as the deposition of both amyloid plaques and neurofibrillary tangles. Studies from our group and others demonstrated, in DS brain, the dysfunction of both proteasome and autophagy degradative systems, coupled with increased oxidative damage. Further, we observed the aberrant increase of mTOR signaling and of its down-stream pathways in both DS brain and in Ts65Dn mice. Based on these findings, we support the ability of intranasal rapamycin treatment (InRapa) to restore mTOR pathway but also to restrain oxidative stress resulting in the decreased accumulation of lipoxidized proteins. By proteomics approach, we were able to identify specific proteins that showed decreased levels of HNE-modification after InRapa treatment compared with vehicle group. Among MS-identified proteins, we found that reduced oxidation of arginase-1 (ARG-1) and protein phosphatase 2A (PP2A) might play a key role in reducing brain damage associated with synaptic transmission failure and tau hyperphosphorylation. InRapa treatment, by reducing ARG-1 protein-bound HNE levels, rescues its enzyme activity and conceivably contribute to the recovery of arginase-regulated functions. Further, it was shown that PP2A inhibition induces tau hyperphosphorylation and spatial memory deficits. Our data suggest that InRapa was able to rescue PP2A activity as suggested by reduced p-tau levels. In summary, considering that mTOR pathway is a central hub of multiple intracellular signaling, we propose that InRapa treatment is able to lower the lipoxidation-mediated damage to proteins, thus representing a valuable therapeutic strategy to reduce the early development of AD pathology in DS population.
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http://dx.doi.org/10.1016/j.redox.2019.101162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859577PMC
May 2019

Proteomics of the acid-soluble fraction of whole and major gland saliva in burning mouth syndrome patients.

Arch Oral Biol 2019 Feb 20;98:148-155. Epub 2018 Nov 20.

Institute of Chemistry of the Molecular Recognition - CNR, L.go F. Vito 1, 00168, Rome, Italy.

Objective: In the present study the salivary proteome of burning mouth syndrome patients and healthy subjects was characterized by a top-down proteomic approach and compared to highlight possible qualitative and quantitative differences that may give suggestions about the causes of this pathology which are still unknown.

Materials And Methods: Resting and stimulated whole saliva, stimulated parotid and submandibular/sublingual saliva samples were collected from burning mouth syndrome patients (n = 16) and age- and gender-matched healthy subjects (n = 14). An equal volume of 0.2% trifluoroacetic acid was added to each sample immediately after collection and the supernatants were analysed by liquid chromatography coupled to electrospray-ionisation mass spectrometry. Proteins and peptides were quantified using a label-free approach measuring the extracted ion current peak areas of the main salivary proteins and peptides.

Results: The quantitation of the main salivary proteins and peptides revealed a higher concentration of cystatin SN in resting saliva of burning mouth syndrome patients with respect to healthy controls and no other conspicuous changes.

Conclusions: The reported data showed that the salivary protein profile was not affected, in composition and relative abundance, by the burning mouth syndrome, except for the cystatin SN, a protein up-regulated in several pathological conditions, that might be considered potentially indicative of the disease.
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http://dx.doi.org/10.1016/j.archoralbio.2018.11.020DOI Listing
February 2019

The extreme hyper-reactivity of Cys94 in lysozyme avoids its amorphous aggregation.

Sci Rep 2018 10 30;8(1):16050. Epub 2018 Oct 30.

Department of Chemical Sciences and Technologies, University of Rome "Tor Vergata", Rome, Italy.

Many proteins provided with disulfide bridges in the native state undergo amorphous irreversible aggregation when these bonds are not formed. Here we show that egg lysozyme displays a clever strategy to prevent this deleterious aggregation during the nascent phase when disulfides are still absent. In fact, when the reduced protein assembles into a molten globule state, its cysteines acquire strong hyper-reactivity towards natural disulfides. The most reactive residue, Cys94, reacts with oxidized glutathione (GSSG) 3000 times faster than an unperturbed protein cysteine. A low pK of its sulfhydryl group (6.6/7.1) and a productive complex with GSSG (K = 0.3 mM), causes a fast glutathionylation of this residue (t = 3 s) and a complete inhibition of the protein aggregation. Other six cysteines display 70 times higher reactivity toward GSSG. The discovery of extreme hyper-reactivity in cysteines only devoted to structural roles opens new research fields for Alzheimer's and Parkinson diseases.
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http://dx.doi.org/10.1038/s41598-018-34439-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207692PMC
October 2018

Protein nitration profile of CD3 lymphocytes from Alzheimer disease patients: Novel hints on immunosenescence and biomarker detection.

Free Radic Biol Med 2018 12 12;129:430-439. Epub 2018 Oct 12.

Department of Biomedical Sciences and Biotechnologies, University of Brescia, Brescia, Italy.

Alzheimer's disease (AD) is a progressive form of dementia characterized by increased production of amyloid-β plaques and hyperphosphorylated tau protein, mitochondrial dysfunction, elevated oxidative stress, reduced protein clearance, among other. Several studies showed systemic modifications of immune and inflammatory systems due, in part, to decreased levels of CD3 lymphocytes in peripheral blood in AD. Considering that oxidative stress, both in the brain and in the periphery, can influence the activation and differentiation of T-cells, we investigated the 3-nitrotyrosine (3-NT) proteome of blood T-cells derived from AD patients compared to non-demented (ND) subjects by using a proteomic approach. 3-NT is a formal protein oxidation and index of nitrosative stress. We identified ten proteins showing increasing levels of 3-NT in CD3 T-cells from AD patients compared with ND subjects. These proteins are involved in energy metabolism, cytoskeletal structure, intracellular signaling, protein folding and turnover, and antioxidant response and provide new insights into the molecular mechanism that impact reduced T-cell differentiation in AD. Our results highlight the role of peripheral oxidative stress in T-cells related to immune-senescence during AD pathology focusing on the specific targets of protein nitration that conceivably can be suitable to further therapies. Further, our data demonstrate common targets of protein nitration between the brain and the periphery, supporting their significance as disease biomarkers.
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http://dx.doi.org/10.1016/j.freeradbiomed.2018.10.414DOI Listing
December 2018

Top-down proteomic profiling of human saliva in multiple sclerosis patients.

J Proteomics 2018 09 4;187:212-222. Epub 2018 Aug 4.

Department of Life and Environmental Sciences, Biomedical Section, University of Cagliari, Monserrato Campus, 09042 Monserrato, Cagliari, Italy.

Multiple sclerosis is a chronic disease of the central nervous system characterized by inflammation, demyelination and neurodegeneration which is of undetermined origin. To date a single diagnostic test of multiple sclerosis does not exists and novel biomarkers are demanded for a more accurate and early diagnosis. In this study, we performed the quantitative analysis of 119 salivary peptides/proteins from 49 multiple sclerosis patients and 54 healthy controls by a mass spectrometry-based top-down proteomic approach. Statistical analysis evidenced different levels on 23 proteins: 8 proteins showed lower levels in multiple sclerosis patients with respect to controls and they were mono- and di-oxidized cystatin SN, mono- and di-oxidized cystatin S1, mono-oxidized cystatin SA and mono-phosphorylated statherin. 15 proteins showed higher levels in multiple sclerosis patients with respect to controls and they were antileukoproteinase, two proteoforms of Prolactin-Inducible Protein, P-C peptide (Fr.1-14, Fr. 26-44, and Fr. 36-44), SV1 fragment of statherin, cystatin SN Des, cystatin SN P → L variant, and cystatin A T → M variant. The differences observed between the salivary proteomic profile of patients suffering from multiple sclerosis and healthy subjects is consistent with the inflammatory condition and altered immune response typical of the pathology. Data are available via ProteomeXchange with identifier PXD009440.

Significance: To date a single diagnostic test of multiple sclerosis does not exist, and diagnosis is based on multiple tests which mainly include the analysis of cerebrospinal fluid. However, the need for lumbar puncture makes the analysis of cerebrospinal fluid impractical for monitoring disease activity and response to treatment. The possible use of saliva as a diagnostic fluid for oral and systemic diseases has been largely investigated, but only marginally in multiple sclerosis compared to other body fluids. Our study demonstrates that the salivary proteome of multiple sclerosis patients differs considerably compared to that of sex and age matched healthy individuals and suggests that some differences might be associated with the different disease-modifying therapy used to treat multiple sclerosis patients.
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http://dx.doi.org/10.1016/j.jprot.2018.07.019DOI Listing
September 2018

Extensive Characterization of the Human Salivary Basic Proline-Rich Protein Family by Top-Down Mass Spectrometry.

J Proteome Res 2018 09 20;17(9):3292-3307. Epub 2018 Aug 20.

Department of Life and Environmental Sciences , University of Cagliari, Cittadella Univ. Monserrato , Monserrato 09042 , Cagliari , Italy.

Human basic proline-rich proteins and basic glycosylated proline-rich proteins, encoded by the polymorphic PRB1-4 genes and expressed only in parotid glands, are the most complex family of adult salivary proteins. The family includes 11 parent peptides/proteins and more than 6 parent glycosylated proteins, but a high number of proteoforms with rather similar structures derive from polymorphisms and post-translational modifications. 55 new components of the family were characterized by top-down liquid chromatography-mass spectrometry and tandem-mass platforms, bringing the total number of proteoforms to 109. The new components comprise the three variants P-H S → A, P-Ko P → S, and P-Ko A → S and several of their naturally occurring proteolytic fragments. The paper represents an updated reference for the peptides included in the heterogeneous family of proteins encoded by PRB1/PRB4. MS data are available via ProteomeXchange with the identifier PXD009813.
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http://dx.doi.org/10.1021/acs.jproteome.8b00444DOI Listing
September 2018

Thymosin fraction 5 re-evaluated after 35 years by high-resolution mass spectrometry.

Expert Opin Biol Ther 2018 07;18(sup1):199-203

a Friedrich-Alexander-University Erlangen-Nuremberg , Institute of Biochemistry , Erlangen , Germany.

Objectives: We reevaluated a lyophilized sample of thymosin fraction 5, stored for 37 years at room temperature, by high-resolution mass spectrometry in terms of stability and yet uncharacterized polypeptides that could be biological important substances.

Methods: A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to molecular characterization of polypeptides present in thymosin fraction 5.

Results: We detected more than 100 monoisotopic masses corresponding to thymosin β4 and truncated forms of ubiquitin, prothymosin α, thymosin β4, and thymosin β9. Additionally, we discovered a new polypeptide present in thymosin fraction 5 and identified it as intact SH3 domain-binding glutamic acid-rich-like protein 3.

Conclusion: In spite of the well-known proteolytic processes inherent to the preparation of thymosin fraction 5, still uncharacterized polypeptides as well as truncated forms of already well-known thymosins are present in fraction 5 after long-term storage. Therefore, continuing characterization of thymosin fraction 5 is even nowadays highly promising.
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http://dx.doi.org/10.1080/14712598.2018.1474196DOI Listing
July 2018

The activity of a mammalian proline-rich peptide against Gram-negative bacteria, including drug-resistant strains, relies on a nonmembranolytic mode of action.

Infect Drug Resist 2018 17;11:969-979. Epub 2018 Jul 17.

Institute for the Chemistry of Molecular Recognition, C.N.R., c/o Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy.

Background: A peptide of 2,733 Da named SP-E, previously isolated from pig saliva and already described for its antifungal activity and absence of toxicity against mammalian cells, is characterized by a high content of proline residues (70% of entire sequence), that confer structural features probably related to peptide activity.

Purpose: The aim of this study was to evaluate the activity of SP-E against Gram-negative bacteria, including drug-resistant clinical isolates.

Methods: SP-E and shorter fragments of the same peptide were tested in vitro against the selected bacteria by colony forming unit assays. Scanning electron microscopy and confocal microscopy were also applied. SP-E potential therapeutic activity was evaluated in vivo in a model of bacterial infection.

Results: SP-E proved to be active against the tested bacteria with EC values in the micro-molar range. Though maintaining antibacterial properties, the shorter peptides showed lower activity in respect to the parental molecule. Kinetics of killing action and nonmembranolytic internalization within and cells strongly suggested a cytosolic mechanism of action involving one or more intracellular molecular targets. A single injection of SP-E exerted a therapeutic effect in larvae infected with .

Conclusion: The biological properties of SP-E strongly back this peptide as a new promising multitasking antimicrobial molecule.
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http://dx.doi.org/10.2147/IDR.S165179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054295PMC
July 2018

Proteomic identification of altered protein O-GlcNAcylation in a triple transgenic mouse model of Alzheimer's disease.

Biochim Biophys Acta Mol Basis Dis 2018 10 18;1864(10):3309-3321. Epub 2018 Jul 18.

Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Rome, Italy. Electronic address:

PET scan analysis demonstrated the early reduction of cerebral glucose metabolism in Alzheimer disease (AD) patients that can make neurons vulnerable to damage via the alteration of the hexosamine biosynthetic pathway (HBP). Defective HBP leads to flawed protein O-GlcNAcylation coupled, by a mutual inverse relationship, with increased protein phosphorylation on Ser/Thr residues. Altered O-GlcNAcylation of Tau and APP have been reported in AD and is closely related with pathology onset and progression. In addition, type 2 diabetes patients show an altered O-GlcNAcylation/phosphorylation that might represent a link between metabolic defects and AD progression. Our study aimed to decipher the specific protein targets of altered O-GlcNAcylation in brain of 12-month-old 3×Tg-AD mice compared with age-matched non-Tg mice. Hence, we analysed the global O-GlcNAc levels, the levels and activity of OGT and OGA, the enzymes controlling its cycling and protein specific O-GlcNAc levels using a bi-dimensional electrophoresis (2DE) approach. Our data demonstrate the alteration of OGT and OGA activation coupled with the decrease of total O-GlcNAcylation levels. Data from proteomics analysis led to the identification of several proteins with reduced O-GlcNAcylation levels, which belong to key pathways involved in the progression of AD such as neuronal structure, protein degradation and glucose metabolism. In parallel, we analysed the O-GlcNAcylation/phosphorylation ratio of IRS1 and AKT, whose alterations may contribute to insulin resistance and reduced glucose uptake. Our findings may contribute to better understand the role of altered protein O-GlcNAcylation profile in AD, by possibly identifying novel mechanisms of disease progression related to glucose hypometabolism.
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http://dx.doi.org/10.1016/j.bbadis.2018.07.017DOI Listing
October 2018

Mass spectrometry characterization of DOTA-Nimotuzumab conjugate as precursor of an innovative β tracer suitable in radio-guided surgery.

J Pharm Biomed Anal 2018 Jul 12;156:8-15. Epub 2018 Mar 12.

Institute of Biochemistry and Clinical Biochemistry, Catholic University, Largo F. Vito 1, 00168, Rome, Italy; Institute of Chemistry of Molecular Recognition, CNR, Largo F. Vito 1, 00168, Rome, Italy.

The aim of the present work has been the mass spectrometry characterization of the Nimotuzumab (NIM) antibody chemically modified with the bifunctional chelating agent para-S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraaza cyclododecanetetraacetic acid (p-SCN-Bn-DOTA). The conjugate, upon labeling with the pure β-emitter Y could represent a promising candidate as radiotracer for an innovative radio-guided surgery (RGS) technique, developed and patented by researchers of our group, which uses a probe system for intraoperative detection of tumor residues exploiting the selective uptake of β-emitting tracers. The results reported in this study show that multiple DOTA molecules bind to lysine residues of both light and heavy chains of the antibody and, probably, some of them are linked to the variable region of antibody. Moreover, the new mass spectrometric analysis highlights the presence of unreacted NIM in the final product. The information obtained by this work is of fundamental importance in the perspective to utilize this conjugate as a radiocompound after its labeling with Y radioisotope. Indeed, the conjugation efficiency and the presence of unreacted NIM affect the specific activity of the final radiotracer which binds specific receptor.
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http://dx.doi.org/10.1016/j.jpba.2018.03.018DOI Listing
July 2018

Testosterone and FSH modulate Sertoli cell extracellular secretion: Proteomic analysis.

Mol Cell Endocrinol 2018 11 25;476:1-7. Epub 2018 Apr 25.

Department of Experimental Medicine, University of Perugia, Perugia, 06100, Italy; Division of Medical Andrology and Endocrinology of Reproduction, University of Perugia and Saint Mary Hospital, Terni, 05100, Italy. Electronic address:

Spermatogenesis is a highly complicated biological process that occurs in the epithelium of the seminiferous tubules. It is regulated by a complex network of endocrine and paracrine factors and by juxtacrine testicular cross-talk. Sertoli cells (SC) play a key role in spermatogenesis due to their production of trophic, differentiation and immune-modulating factors, but many of the molecular pathways of SC action remain controversial and unclear. Over the last two decades, research has focused on extracellular vesicles as an important mechanism of intercellular communication. The aim of this study was to investigate the presence of extracellular vesicles (EVs) in SC and the modulation of their content in SC after FSH and testosterone stimulation. Highly purified porcine pre-pubertal Sertoli cells were isolated according to previously established methods. After 48 h of culture with FSH or FSH + testosterone stimulation, we identified sertolian EVs containing specific mRNAs. Proteomic analysis of EVs content identified 29 proteins under non-stimulatory conditions, most of which were related to receptor binding activity. FSH stimulation induced increases in inhibin-alpha, inhibin-beta, plakoglobin, haptoglobin, D-3-phosphoglycerate dehydrogenase and sodium/potassium-transporting ATPase in sertolian EVs. Testosterone stimulation enhanced the abundance of inhibin-alpha, inhibin-beta, tissue-type plasminogen activator, epidermal growth factor-like protein 8, elongating factor 1-gamma and D-3-phosphoglycerate dehydrogenase. These results are likely to help determine the unknown molecular secretion of Sertoli cells.
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http://dx.doi.org/10.1016/j.mce.2018.04.001DOI Listing
November 2018

Semen Proteomics Reveals the Impact of Enterococcus faecalis on male Fertility.

Protein Pept Lett 2018 ;25(5):472-477

International Scientific Institute "Paul VI", Catholic University, Rome, Italy.

Background: Infectious etiologies contribute to 15% of male factor infertility. Enterococcus faecalis (E. faecalis) is commonly identified in semen culture of infertile men and it is associated with significantly poorer semen quality.

Objective: Aim of this study was to identify new seminal biomarkers for the male tract infection by E. faecalis, using proteomic profiling, in order to understand the effect of E. faecalis on the physiopathology of male reproduction.

Methods: We included in the study ten patients seeking medical care for primary infertility with prostate-vesicular-epidydimitis and with microbiological analysis on semen and/or prostatic secretions positive for E. faecalis. Ten fertile men have been enrolled as a control group in the protocol. An aliquot of each seminal plasma was subjected to an in-solution digestion protocol and analyzed using an Ultimate 3000 RSLCnano HPLC apparatus coupled to a LTQ Orbitrap Elite mass spectrometer.

Results: Eight proteins have not been identified in the group of controls and have been observed in a remarkable proportion of patients, mainly involved in immune system activity (CD177, Swiprosin-1 and 2-oxoglutarate dehydrogenase). Arylsulfatase has been identified in the group of controls and was absent in all patients with infection. Three proteins (TIMP-1, WFDC domain protein 2 and Carboxypeptidase E) have been observed significantly different in patients versus controls, mainly related with inflammation.

Conclusions: This is the first application of MS-based proteomics aimed to reveal an array of proteins in the seminal plasma and reflecting the effect of the infection by E. faecalis on semen composition.
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http://dx.doi.org/10.2174/0929866525666180412161818DOI Listing
August 2018