on Analytical chemistry
by Illiano A, Arpino V, Pinto G, Berti A, Verdoliva V, Peluso G, Pucci P, Amoresano A
Knowledge of the nature of biofluids at a crime scene is just as important as DNA test to link the nature of the biofluid, the criminal act, and the dynamics of the crime. Identification of methods currently used for each biological fluid (blood, semen, saliva, urine) suffer from several limitations including instability of assayed biomolecules, and low selectivity and specificity; as an example of the latter issue, it is not possible to discriminate between alpha-amylase 1 (present in saliva) and alpha-amylase 2 (present in semen and vaginal secretion. In this context, the aim of the work has been to provide a predictive protein signature characteristic of each biofluid by the recognition of specific peptides unique for each protein in a single analysis. A panel of four protein biomarkers for blood, four for saliva, five for semen, and two for urine has been monitored has been monitored by using a single multiple reaction monitoring (MRM)-based method targeting concomitantly 46 different peptides. Then, The optimized method allows four biological matrices to be identified when present on their own or in 50:50 mixture with another biofluid. Finally, a valid strategy combining both DNA analysis and liquid chromatographic-tandem mass spectrometric multiple reaction monitoring (LC-MS-MRM) identification of biofluids on the same sample has been demonstrated to be particularly effective in forensic investigation of real trace evidence collected at a crime scene.
on Biology of the cell
by Armentano MF, Caterino M, Miglionico R, Ostuni A, Pace MC, Cozzolino F, Monti M, Milella L, Carmosino M, Pucci P, Bisaccia F
Up-regulated Gene clone 7 (URG7) is an ER resident protein, whose expression is upregulated in the presence of hepatitis B virus X antigen (HBxAg) during HBV infection. In virus-infected hepatocytes, URG7 shows an anti-apoptotic activity due to the PI3K/AKT signaling activation, does not seem to have tumorigenic properties, but it appears to promote the development and progression of fibrosis. However, the molecular mechanisms underlying URG7 activity remain largely unknown.
on Free radical biology & medicine
by Butturini E, Cozzolino F, Boriero D, Carcereri de Prati A, Monti M, Rossin M, Canetti D, Cellini B, Pucci P, Mariotto S
STAT1 and STAT3 are two transcription factors involved in a lot of cellular functions such as immune response, proliferation, apoptosis, and cell survival. A number of literature evidences described a yin-yang relationship between activation of STAT1 and STAT3 in neurodegenerative disorders where STAT1 exerts a pro-apoptotic effect whereas STAT3 shows neuroprotective properties through the inhibition of apoptosis. Although the role of oxidative-stress in the pathogenesis of neurodegeneration is clearly described, its influence in the regulation of these pathways is poorly understood. Herein, we demonstrate that HO rapidly induces phosphorylation of STAT1 whereas it is not able to influence phosphorylation of STAT3 in mouse microglia BV2 cells. The analysis of the molecular mechanism of STATs signaling reveals that HO induces S-glutathionylation of both STAT1 and STAT3. The same post-translational event exerts an opposing role in the regulation of STAT1 and STAT3 signaling. These data not only confirm redox sensibility of STAT3 signaling but also reveal for the first time that STAT1 is susceptible to redox regulation. A deep study of the molecular mechanism of STAT1 redox regulation, identifies Cys324 and Cys492 as the main targets of S-glutathionylation and confirms that S-glutathionylation does not impair JAK2 mediated STAT1 tyrosine phosphorylation. These results demonstrate that both phosphorylation and glutathionylation contribute to activation of STAT1 during oxidative stress and underline that the same post-translation event exerts an opposing role in the regulation of STAT1 and STAT3 signaling in microglia cells.
on Pharmacological research
by Prevete N, Liotti F, Amoresano A, Pucci P, de Paulis A, Melillo RM
Inflammation is considered an enabling feature of cancer. Besides the persistence of inflammatory stimuli, also defective mechanisms of resolution can lead to chronic inflammation. Inflammation resolution is an active process controlled by lipidic specialized pro-resolving mediators (SPMs), derived from ω-3 or ω-6 essential polyunsaturated fatty acids (PUFA) through the activity of lipoxygenases (ALOX5 and 15). Thus, a lack or defect in resolution mechanisms may affect cancer development and progression by prolonging inflammation. Components of pro-resolving pathways (PUFA, enzymes, or SPMs) have been reported to modulate various cancer features by affecting both epithelial cells and cancer-associated stroma. Here, we will review the most important mechanisms by which SPMs, ω-3/6 PUFA, and ALOXs affect cancer biology, paying particular attention to their role in the inhibition of inflammation and angiogenesis, two of the most important hallmarks of cancer. The collection of these results may suggest novel perspectives in cancer management based on the modulation of lipid metabolism and the production of SPMs.
on Scientific reports
by Salvia R, Grossi G, Amoresano A, Scieuzo C, Nardiello M, Giangrande C, Laurenzana I, Ruggieri V, Bufo SA, Vinson SB, Carmosino M, Neunemann D, Vogel H, Pucci P, Falabella P
Toxoneuron nigriceps (Hymenoptera, Braconidae) is an endophagous parasitoid of the larval stages of the tobacco budworm, Heliothis virescens (Lepidoptera, Noctuidae). The bracovirus associated with this wasp (TnBV) is currently being studied. Several genes expressed in parasitised host larvae have been isolated and their possible roles partly elucidated. TnBVank1 encodes an ankyrin motif protein similar to insect and mammalian IκB, an inhibitor of the transcription nuclear factor κB (NF-κB). Here we show that, when TnBVank1 was stably expressed in polyclonal Drosophila S2 cells, apoptosis is induced. Furthermore, we observed the same effects in haemocytes of H. virescens larvae, after TnBVank1 in vivo transient transfection, and in haemocytes of parasitised larvae. Coimmunoprecipitation experiments showed that TnBVANK1 binds to ALG-2 interacting protein X (Alix/AIP1), an interactor of apoptosis-linked gene protein 2 (ALG-2). Using double-immunofluorescence labeling, we observed the potential colocalization of TnBVANK1 and Alix proteins in the cytoplasm of polyclonal S2 cells. When Alix was silenced by RNA interference, TnBVANK1 was no longer able to cause apoptosis in both S2 cells and H. virescens haemocytes. Collectively, these results indicate that TnBVANK1 induces apoptosis by interacting with Alix, suggesting a role of TnBVANK1 in the suppression of host immune response observed after parasitisation by T. nigriceps.
on PloS one
by Fontanarosa C, Pane F, Sepe N, Pinto G, Trifuoggi M, Squillace M, Errico F, Usiello A, Pucci P, Amoresano A
Several studies have suggested that free d-Asp has a crucial role in N-methyl d-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free d-Asp, l-Asp and N-methyl d-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing l-Asp, d-Asp and N-methyl d-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75-110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for d-Asp, 0.46 pg/μl for l-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for d-Asp, 1.41 pg/μl for l-Asp and 1.64 pg/μl for NMDA, respectively. Different concentration levels were used for constructing the calibration curves which showed good linearity. The validated method was then successfully applied to the simultaneous detection of d-Asp, l-Asp and NMDA in mouse brain tissues. The concurrent, sensitive, fast, and reproducible measurement of these metabolites in brain tissues will be useful to correlate the amount of free d-Asp with relevant neurological processes, making the LC-MS/MS MRM method well suited, not only for research work but also for clinical analyses.
on Scientific reports
by Iaconis D, Monti M, Renda M, van Koppen A, Tammaro R, Chiaravalli M, Cozzolino F, Pignata P, Crina C, Pucci P, Boletta A, Belcastro V, Giles RH, Maria Surace E, Gallo S, Pende M, Franco B
Protein synthesis is traditionally associated with specific cytoplasmic compartments. We now show that OFD1, a centrosomal/basal body protein, interacts with components of the Preinitiation complex of translation (PIC) and of the eukaryotic Initiation Factor (eIF)4F complex and modulates the translation of specific mRNA targets in the kidney. We demonstrate that OFD1 cooperates with the mRNA binding protein Bicc1 to functionally control the protein synthesis machinery at the centrosome where also the PIC and eIF4F components were shown to localize in mammalian cells. Interestingly, Ofd1 and Bicc1 are both involved in renal cystogenesis and selected targets were shown to accumulate in two models of inherited renal cystic disease. Our results suggest a possible role for the centrosome as a specialized station to modulate translation for specific functions of the nearby ciliary structures and may provide functional clues for the understanding of renal cystic disease.
on Brain : a journal of neurology
by Zollo M, Ahmed M, Ferrucci V, Salpietro V, Asadzadeh F, Carotenuto M, Maroofian R, Al-Amri A, Singh R, Scognamiglio I, Mojarrad M, Musella L, Duilio A, Di Somma A, Karaca E, Rajab A, Al-Khayat A, Mohan Mohapatra T, Eslahi A, Ashrafzadeh F, Rawlins LE, Prasad R, Gupta R, Kumari P, Srivastava M, Cozzolino F, Kumar Rai S, Monti M, Harlalka GV, Simpson MA, Rich P, Al-Salmi F, Patton MA, Chioza BA, Efthymiou S, Granata F, Di Rosa G, Wiethoff S, Borgione E, Scuderi C, Mankad K, Hanna MG, Pucci P, Houlden H, Lupski JR, Crosby AH, Baple EL
PRUNE is a member of the DHH (Asp-His-His) phosphoesterase protein superfamily of molecules important for cell motility, and implicated in cancer progression. Here we investigated multiple families from Oman, India, Iran and Italy with individuals affected by a new autosomal recessive neurodevelopmental and degenerative disorder in which the cardinal features include primary microcephaly and profound global developmental delay. Our genetic studies identified biallelic mutations of PRUNE1 as responsible. Our functional assays of disease-associated variant alleles revealed impaired microtubule polymerization, as well as cell migration and proliferation properties, of mutant PRUNE. Additionally, our studies also highlight a potential new role for PRUNE during microtubule polymerization, which is essential for the cytoskeletal rearrangements that occur during cellular division and proliferation. Together these studies define PRUNE as a molecule fundamental for normal human cortical development and define cellular and clinical consequences associated with PRUNE mutation.
by Prevete N, Liotti F, Illiano A, Amoresano A, Pucci P, de Paulis A, Melillo RM
Chronic inflammation can result from inadequate engagement of resolution mechanisms, mainly accomplished by specialized pro-resolving mediators (SPMs) arising from the metabolic activity of lipoxygenases (ALOX5/15) on ω-6 or ω-3 essential polyunsaturated fatty acids (PUFA). We previously demonstrated that formyl peptide receptor 1 (FPR1) suppresses gastric cancer (GC) by inhibiting its inflammatory/angiogenic potential. In this study, we asked whether FPR1 exploits inflammation resolution pathways to suppress GC angiogenesis and growth. Here, we demonstrate that genetic or pharmacologic modulation of FPR1 in GC cells regulated ALOX5/15 expression and production of the SPMs Resolvin D1 (RvD1) and Lipoxin B4 (LXB4). SPM treatment of GC cells abated their angiogenic potential. Genetic deletion of ALOX15 or of the RvD1 receptor GPR32 increased the angiogenic and tumorigenic activity of GC cells thereby mimicking FPR1 loss. Deletion/inhibition of ALOX5/15 or GPR32 blocked FPR1-mediated anti-angiogenic activities, indicating that ALOX5/15 and GPR32 are required for FPR1's pro-resolving action. An ω-3- or ω-6-enriched diet enforced SPM endogenous production in mice and inhibited growth of shFPR1 GC xenografts by suppressing their angiogenic activity. These data implicate that FPR1 and/or pro-resolving pathway components might be used as risk/prognostic markers for GC; ω-6/3-enriched diets, and targeting FPR1 or SPM machinery may be exploited for GC management.
on International journal of molecular sciences
by Esposito MV, Nunziato M, Starnone F, Telese A, Calabrese A, D'Aiuto G, Pucci P, D'Aiuto M, Baralle F, D'Argenio V, Salvatore F
About 10% of all breast cancers arise from hereditary mutations that increase the risk of breast and ovarian cancers; and about 25% of these are associated with the BRCA1 or BRCA2 genes. The identification of BRCA1/BRCA2 mutations can enable physicians to better tailor the clinical management of patients; and to initiate preventive measures in healthy carriers. The pathophysiological significance of newly identified variants poses challenges for genetic counseling. We characterized a new BRCA1 variant discovered in a breast cancer patient during BRCA1/2 screening by next-generation sequencing. Bioinformatic predictions; indicating that the variant is probably pathogenetic; were verified using retro-transcription of the patient's RNA followed by PCR amplifications performed on the resulting cDNA. The variant causes the loss of a canonic donor splice site at position +2 in BRCA1 intron 21; and consequently the partial retention of 156 bp of intron 21 in the patient's transcript; which demonstrates that this novel BRCA1 mutation plays a pathogenetic role in breast cancer. These findings enabled us to initiate appropriate counseling and to tailor the clinical management of this family. Lastly; these data reinforce the importance of studying the effects of sequence variants at the RNA level to verify their potential role in disease onset.