Industrial Research and Technology Transfer Laboratory accredited by Emilia Romagna Region (DGR 1213/2007) with Deliberation n.GPG/2012/119 of February 6, 2012.
Industrial Research and Technology Transfer Laboratory accredited by Emilia Romagna Region (DGR 1213/2007) with Deliberation n.GPG/2012/119 of February 6, 2012.
The research group has a well-established experience in the study of blood coagulation factors, including i) the analysis of intracellular and plasma expression levels, ii) the characterization of natural and engineered protein variants by expression studies in eukaryotic cells, and iii) their specific transcriptional, post-transcriptional and translational modulation.
The scientific activity of the group is demonstrated by several publications in international peer-reviewed journals.
Our activity is focused on the investigation of the efficiency of blood coagulation cascade in plasma samples from patients affected by coagulation disorders, or in deficient plasma supplemented with recombinant proteins of interest. To this purpose in our laboratory we have optimized small scale functional assays with specific fluorogenic substrates that permit us to monitor the initiation (generation of activated factor X) and amplification (generation of thrombin) phase of coagulation.
These approaches have been used for the biochemical characterization of natural and engineered protein variants of coagulation factors produced by recombinant technologies and expression in eukaryotic cells.
The haemostatic process is tightly regulated through a balance of anti- and pro-coagulant protein components, such as factor VII, factor IX, factor X and thrombin.
Alterations in plasma levels of these proteins, due to genetic or acquired factors (including pharmacological treatments) cause haemorrhagic or pro-thrombotic events whose clinical relevance depends on their relative quantitative variation. The characterization of altered protein levels of specific factors and their effects on physiological processes can be obtained through functional studies performed on plasma samples. Together with traditional coagulation assays, we have optimized functional assays that permit us to evaluate the efficiency of the blood coagulation cascade at different levels, from the initiation to the amplification phase, including the generation of thrombin, which is the final effector of the whole process. These small scale assays take advantage of specific fluorogenic substrates. Moreover, we have developed binding assays to evaluate the interaction of coagulation factors with other molecules or complexes.
Data obtained by these methods can give information about the functional efficacy of blood coagulation both in humans and animal models, and can also be exploited for studies on biocompatibility of those biomaterials used for medical devices, such as dialysis systems or filters, which can come in contact with blood. Indeed, these interactions could lead to the activation or inhibition of coagulation cascade, or cause the depletion of specific proteins or macromolecular complexes, thus potentially leading to adverse events of physiopathological relevance. The knowledge of these aspects and multiple interactions pave the way for the design of the most suitable and compatible biomaterials or devices.
Functional assays
In our laboratory we have optimized small scale functional assays that enable us to monitor the initiation (generation of activated factor X) and amplification (generation of thrombin) phase of coagulation, in order to evaluate their efficiency in healthy patients or patients who experienced treatment/processing with devices of different nature (biocompatibility data). These assays are highly specific and sensitive. Analysis is performed on patient plasma samples, which are compared with pooled normal plasma used as reference. Moreover, the small scale features of these assays ensure the usage of microvolumes of samples and a high number of replicates, thus allowing highly consistent and reproducible data.
This Service also supplies with software-based analysis (GraphPad Prism) of the main parameters underlying the activity or coagulant capacity of the plasma samples tested.
ELISA and binding assays
Quantitative analysis through immunoenzymatic assays is available for specific coagulation factors.
Moreover, we have optimized ELISA-based binding assays on 96-well microplates for the evaluation of interactions between coagulation factors and other molecules or complexes.
This Service also supplies with software-based analysis (GraphPad Prism) of the binding constants describing the binding capacity of the tested molecules.
Mono- and two-dimensional electrophoresis
Mono-dimensional electrophoresis on constant or gradient polyacrilamide gel in denaturing and/or reducing conditions allow proteins to be separated on the basis of their molecular weight. Complex mixtures such as plasma, biological fluids, cellular extracts or supernatants, or purified proteins can be qualitatively or semi-quantitatively analyzed by this technique.
The Service is also implemented by other related techniques:
The relevant instrumentations include:
Molecular Interactions, Biomarkers and Delivery facility
Laboratorio per le Tecnologie delle Terapie Avanzate (LTTA)
Area Polo Chimico Biomedico ‘CUBO’ – second floor
Via Fossato di Mortara, 70 - 44121 Ferrara
+39 0532 455859 - 455865
Prof. Mirko Pinotti
pnm@unife.it
+39 0532 974424
Dr Alessio Branchini (Ph.D.)
brnlss@unife.it
+39 0532 974421
Dr Alessio Branchini
Dr Marcello Baroni
| Malin Bern, Jeannette Nilsen, Mattia Ferrarese, Kine M. K. Sand, Torleif T. Gjølberg, Heidrun E. Lode, Robert J. Davidson, Rodney M. Camire, Espen S. Bækkevold, Stian Foss, Algirdas Grevys, Bjørn Dalhus, John Wilson, Lene S. Høydahl, Gregory J. Christianson, Derry C. Roopenian, Tilman Schlothauer, Terje E. Michaelsen, Morten C. Moe, Silvia Lombardi, Mirko Pinotti, Inger Sandlie, Alessio Branchini and Jan Terje Andersen. An engineered human albumin enhances half-life and transmucosal delivery when fused to protein-based biologics. Science Translational Medicine 14 Oct 2020: Vol. 12, Issue 565, eabb0580. DOI: 10.1126/scitranslmed.abb0580 |
| Andersen E, Chollet ME, Baroni M, Pinotti M, Bernardi F, Skarpen E, Sandset PM, Skretting G. The effect of the chemical chaperone 4-phenylbutyrate on secretion and activity of the p.Q160R missense variant of coagulation factor FVII. Cell Biosci. 2019 Aug 27;9:69. doi: 10.1186/s13578-019-0333-8. PubMed |
| Marchi S, Corricelli M, Branchini A, Vitto VAM, Missiroli S, Morciano G, Perrone M, Ferrarese M, Giorgi C, Pinotti M, Galluzzi L, Kroemer G, Pinton P. Akt-mediated phosphorylation of MICU1 regulates mitochondrial Ca2+ levels and tumor growth. EMBO J. 2019 Jan 15;38(2):e99435. doi: 10.15252/embj.201899435. PMCID: PMC6331721. PubMed |
| Donadon I, McVey JH, Garagiola I, Branchini A, Mortarino M, Peyvandi F, Bernardi F, Pinotti M. Clustered F8 missense mutations cause hemophilia A by combined alteration of splicing and protein biosynthesis and activity. Haematologica. 2018 Feb;103(2):344-350. doi: 10.3324/haematol.2017.178327. PubMed |
| Barbon E, Pignani S, Branchini A, Bernardi F, Pinotti M, Bovolenta M (2016) An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes. Sci Rep 6:28304. PubMed |
| Branchini A, Ferrarese M, Lombardi S, Mari R, Bernardi F, Pinotti M (2016) Differential functional readthrough over homozygous nonsense mutations contributes to the bleeding phenotype in coagulation factor VII deficiency. J. Thromb. Haemost. 14:1994-2000. PubMed |
| Branchini A, Baroni M, Burini F, Puzzo F, Nicolosi F, Mari R, Gemmati D, Bernardi F, Pinotti M (2015) The carboxyl-terminal region is NOT essential for secreted and functional levels of coagulation factor X. J. Thromb. Haemost. 13:1468-74. PubMed |
| Branchini A, Baroni M, Pfeiffer C, Batorova A, Giansily-Blaizot M, Schved JF, Mariani G, Bernardi F, Pinotti M (2014) Coagulation factor VII variants resistant to inhibitory antibodies. Thromb. Haemost. 112:972-80. PubMed |
| Balestra D, Faella A, Margaritis P, Cavallari N, Pagani F, Bernardi F, Arruda VR, Pinotti M (2014) An engineered U1 small nuclear RNA rescues splicing defective coagulation F7 gene expression in mice. J. Thromb. Haemost. 12:177-85. PubMed |
| Branchini A, Campioni M, Mazzucconi MG, Biondo F, Mari R, Bicocchi MP, Bernardi F, Pinotti M (2013) Replacement of the Y450 (c234) phenyl ring in the carboxyl-terminal region of coagulation factor IX causes pleiotropic effects on secretion and enzyme activity. FEBS Lett. 587:3249-53. PubMed |
| Olivieri O, Martinelli N, Baroni M, Branchini A, Girelli D, Friso S, Pizzolo F, Bernardi F (2013) Factor II activity is similarly increased in patients with elevated apolipoprotein CIII and in carriers of the factor II 20210A allele. J Am Heart Assoc 2:e000440. PubMed |
| Fernandez Alanis E, Pinotti M, Dal Mas A, Balestra D, Cavallari N, Rogalska ME, Bernardi F, Pagani F (2012) An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects. Hum. Mol. Genet. 21:2389-98. PubMed |
| Pinotti M, Caruso P, Canella A, Campioni M, Tagariello G, Castaman G, Giacomelli S, Belvini D, Bernardi F (2012) Ribosome readthrough accounts for secreted full-length factor IX in hemophilia B patients with nonsense mutations. Hum. Mutat. 33:1373-6. PubMed |
| Branchini A, Rizzotto L, Mariani G, Napolitano M, Lapecorella M, Giansily-Blaizot M, Mari R, Canella A, Pinotti M, Bernardi F (2012) Natural and engineered carboxy-terminal variants: decreased secretion and gain-of-function result in asymptomatic coagulation factor VII deficiency. Haematologica 97:705-9. PubMed |
| Pinotti M, Bernardi F, Dal Mas A, Pagani F (2011) RNA-based therapeutic approaches for coagulation factor deficiencies. J. Thromb. Haemost. 9:2143-52. PubMed |
| Casari C, Pinotti M, Lancellotti S, Adinolfi E, Casonato A, De Cristofaro R, Bernardi F (2010) The dominant-negative von Willebrand factor gene deletion p.P1127_C1948delinsR: molecular mechanism and modulation. Blood 116:5371-6. PubMed |
| Baroni M, Pavani G, Marescotti D, Kaabache T, Borgel D, Gandrille S, Marchetti G, Legnani C, D'Angelo A, Pinotti M, Bernardi F (2010) Membrane binding and anticoagulant properties of protein S natural variants. Thromb. Res. 125:e33-9. PubMed |
| Baroni M, Pizzirani C, Pinotti M, Ferrari D, Adinolfi E, Calzavarini S, Caruso P, Bernardi F, Di Virgilio F (2007) Stimulation of P2 (P2X7) receptors in human dendritic cells induces the release of tissue factor-bearing microparticles. FASEB J. 21:1926-33. PubMed |
| Code no. | Brief description of the services we offer | Price (€ per hour) |
| Preliminary consultation on the experimental design and/or sample preparation; during the consultation a quote can be prepared on the overall cost of the analyses required. The Service offers consultations for specific projects, and quotes are provided free of charge according to the needs of the user. Please contact us for individual requirements not shown in the price list, since the packages offered can be tailored to requirements outside the standard service. For routine analysis prices can be agreed (subscription may be possible). |
0.00 | |
| IM-SC-01 | Functional assays with fluorogenic substrates on plasma samples: Functional assays on patients’ plasma samples are performed by monitoring fluorescence emission as a function of time after addition of fluorogenic substrates specific for the activated factor X or thrombin. Reference samples and controls within the assays are represented by pooled normal plasma and/or commercially-available deficient plasma. The minimal volume of plasma requested for a single reaction is 10 µl. Replicates need additional volume(s). Plasma samples must not be thawed and have to be shipped on dry ice. This Service supplies with software-based analysis (GraphPad Prism) of the main parameters underlying the activity or coagulant capacity of the plasma samples tested. |
Please ask for a quote |
| IM-SC-02 | Quantitative analysis of coagulation factors by ELISA: For specific coagulation factors, the quantitative analysis is available, through immunoenzymatic assays. Dilutions of pooled normal plasma or known concentrations of purified proteins are used as reference. The minimal volume of plasma requested for a single reaction is 10 µl. Replicates need additional volume(s). Plasma samples must not be thawed and have to be shipped on dry ice. This Service supplies with software-based quantitative analysis (GraphPad Prism) of protein concentrations in samples. |
Please ask for a quote |
| IM-SC-03 | Binding studies: Evaluation of the interaction of coated or soluble coagulation factors with other molecules or complexes is available by binding studies on 96-well microplates. Known concentrations of purified proteins are used as reference. Samples must not be thawed and have to be shipped on dry ice. This Service supplies with software-based analysis (GraphPad Prism) of the binding constants describing the binding capacity of the tested molecules. This Service supplies with software-based analysis (GraphPad Prism) of the binding constants describing the binding capacity of the tested molecules. |
Please ask for a quote |
| IM-BD-01 | Mono or bi-dimensional electrophoresis: Polyacrylamide mono- or bi-dimensional gel electrophoresis in denaturing and/or reducing conditions for the separation of protein samples. Complex mixtures such as plasma, biological fluids, cellular extracts or supernatants, or purified proteins can be qualitatively or semi-quantitatively analyzed. The Service is implemented by other related techniques: Staining: gel(s) can be stained with traditional staining methods (silver or Coomassie blue staining) or fluorescent dyes. Band densitometry is also available. Blotting: gel(s) can be transferred to nitrocellulose membranes, and then returned to the customer. Western blotting: for known proteins, expression analysis is available by exploiting the western blotting technique |
Please ask for a quote |
Some of the services/products offered may be provided with the help of other services of the LTTA Laboratory.
Each service includes a description of the procedure and supply of images for scientific publication purposes.
Prices are shown without VAT and are subjected to change according to the cost of consumables necessary (every 12 months).
The innovative AlphaScreen® and the numerous Real-Time PCR applications represent key technologies of our core facility, which make it not only competitive, but also constantly expanding.
AlphaScreen® is the ideal detection technology which can simplify scheduling issues in screening the effects of bioactive molecules in terms of activity and biomolecular interactions. The approach relies on the use of donor (photosensitizer) and acceptor (chemiluminescer) beads, which are coated with proteins whose bioconjugation you intend to analyse. A fluorescent signal is produced when the AlphaScreenTM acceptor and donor beads are brought into proximity by a molecular interaction occurring between the binding partners captured on the beads.
Specifically, laser excitation of donor beads at 680 nm causes ambient oxygen to be converted to the singlet state by photosensitizers. The acceptor beads, on the other hand, contain a chemiluminescent agent, often a thioxene derivative, that reacts with the singlet oxygen, generating chemiluminescence at 370 nm. This energy is immediately transferred to the fluorophores contained in the same acceptor bead, which shifts the emission wavelength to 520-620 nm. The half-life of 0.3 seconds allows detection of fluorescence in time-resolved mode. The detection sensitivity is in the order of attomoles.
Real-time quantitative PCR allows the sensitive, specific and reproducible quantification of a target nucleic acid sequence in a biological sample in real time.
Real-Time PCR instruments not only act as thermal cyclers, but also excite the fluorophores contained in the samples and record the fluorescence in ‘real-time’ as the samples pass photodetection diodes within the instrument. A software acquires the emission spectrum form each fluorophore individually for the duration of the PCR and converts the variation in the reporter fluorescence to a real-time representation of the amplification kinetics.
Fluorescence is generated during PCR by different chemical reactions according to the binding of fluorescent dyes that intercalate in the DNA double helix, such as SYBR Green, or the hybridisation of specific probes. We use Real-Time PCR in:
TaqMan® Real-Time PCR
This technology is widely used in order to measure the expression level of a target sequence. A TaqMan probe is an oligonucleotide complementary to the target sequence to be amplified. The non-extendible hydrolysis (e.g. TaqMan®) probe bears a fluorophore at each end. In the intact probe, the fluorescence generated by the reporter dye in 5’ is quenched by the second fluorophore in 3’. Probe cleavage by the 5’-3’ exonuclease activity of the DNA polymerase during PCR extension releases the reporter and quencher moieties, then fluorescence is emitted and recorded. Unbound probes are not hydrolyzed and consequently, fluorescence is proportional to the quantity of the target product present.
Allelic Discrimination
This is a multiplexed assay (more than one probe per sample) which detects variants of a single nucleic acid sequence. The presence of two TaqMan® MGB (minor groove binder) probes in each reaction, both targeting the same SNP (single-nucleic polymorphism) site, one of them perfectly complementary to the wild type allele and the other to the mutated allele, allows genotyping of the two variants of a single-nucleic polymorphism site in a target sequence. The genotype is defined according to the relative intensity emitted by the two fluorophores.
High resolution melting (HRM)
This technique is based on the amplification of a gene of interest through real-time PCR and subsequent analysis of denaturation kinetic of the amplicons. HRM enables the different DNA sequences to be identified by melting curve analysis. Amplicons are denatured in the presence of an intercalating fluorophore that permits total saturation of DNA without inhibiting the PCR reaction. Saturation prevents relocation of fluorophores during the melting phase. Each duplex (homoduplex or heteroduplex) will exhibit a characteristic melting point in the denaturation phase. HRM records the change in the fluorescence that occurs during the dissociation stage from double strand DNA to single strand DNA.
High Resolution Melting (HRM) enables genomic researchers to detect and discriminate single bases mutations, insertions, deletions, base pair substitutions, polymorphisms and epigenetic differences in double-stranded DNA samples.
The research group has acquired a great expertise in the study of genetic markers and drug response specific cellular markers and in measuring biologically active molecules.
The scientific level is well documented by the numerous publications in international scientific journals.
The research area have been focused on the responses of endocrine and endocrine-related solid tumours to new molecules derived from scientific research.
The Biomarkers Facility is specifically interested in the identification and characterization of biomarkers correlated to the activity of endocrine solid tumours, with possible applications to other medical fields.
A) Tests on CELL CULTURES
Maintenance and expansion of immortalised cell lines and subsequent assays with bioactive molecules:
B) Tests on primary human tissue cultures
Cell culture from tissue explants and subsequent assays with bioactive molecules. We are experienced with the following human tissue:
C) Cell parameter measurements
D) GENOTYPE-PHENOTYPE CORRELATION STUDIES
E) MULTI-MODE PLATE READING
Intensity of fluorescence, luminescence, absorbency (UV, VIS), AlphaScreen technology
F) PLATE-READING IN REAL-TIME PCR USING DIFFERENT TECHNOLOGIES
Services may be modified in accordance with the requirements of individual users.
The qualifying instruments available in our facility are:
Laminar flow hood, in order to maintain the sterility necessary for cell culture;
VERITI 96-well Thermal Cycler, 7900 HT Real Time PCR systems, GelDoc-It UVP gel documentation system and 3130 Genetic Analyzer, for analyses of genetic markers;
EnVision Xcite Multilabel Plate Reader, suitable for non-isotopic detection technologies, such as fluorescence, luminescence or absorbance (UV, VIS) measurements, and for innovative AlphaScreen® technology, in order to study signal transduction pathways.
Biomarkers facility
Laboratorio per le Tecnologie delle Terapie Avanzate (LTTA)
University hospital of Ferrara, Division of Endocrinology – 1E2
Via Aldo Moro 8 - 44124 Cona (Ferrara)
+39 0532 236682
Prof. Maria Chiara Zatelli
ztlmch@unife.it
+39 0532 236682
Dr Giulia Bresciani (Ph.D.)
giulia.bresciani@unife.it
+39 0532 239615
| Falletta S, Partelli S, Rubini C, Nann D, Doria A, Marinoni I, Polenta V, Di Pasquale C, Degli Uberti E, Perren A, Falconi M, Zatelli MC (2016) mTOR inhibitors response and mTOR pathway in pancreatic neuroendocrine tumors. Endocr. Relat. Cancer 23:883-891. PubMed |
| Martini C, Zanchetta E, Di Ruvo M, Nalesso A, Battocchio M, Gentilin E, Degli Uberti E, Vettor R, Zatelli MC (2016) Cushing in a Leaf: Endocrine Disruption From a Natural Remedy. J. Clin. Endocrinol. Metab. 101:3054-60. PubMed |
| Gentilin E, Di Pasquale C, Gagliano T, Tagliati F, Benfini K, Ambrosio MR, Bondanelli M, degli Uberti EC, Zatelli MC (2016) Protein Kinase C Delta restrains growth in ACTH-secreting pituitary adenoma cells. Mol. Cell. Endocrinol. 419:252-8. PubMed |
| Beckers A, Lodish MB, Trivellin G, Rostomyan L, Lee M, Faucz FR, Yuan B, Choong CS, Caberg JH, Verrua E, Naves LA, Cheetham TD, Young J, Lysy PA, Petrossians P, Cotterill A, Shah NS, Metzger D, Castermans E, Ambrosio MR, Villa C, Strebkova N, Mazerkina N, Gaillard S, Barra GB, Casulari LA, Neggers SJ, Salvatori R, Jaffrain-Rea ML, Zacharin M, Santamaria BL, Zacharieva S, Lim EM, Mantovani G, Zatelli MC, Collins MT, Bonneville JF, Quezado M, Chittiboina P, Oldfield EH, Bours V, Liu P, W de Herder W, Pellegata N, Lupski JR, Daly AF, Stratakis CA (2015) X-linked acrogigantism syndrome: clinical profile and therapeutic responses. Endocr. Relat. Cancer 22:353-67. PubMed |
| Molè D, Gentilin E, Ibañez-Costa A, Gagliano T, Gahete MD, Tagliati F, Rossi R, Pelizzo MR, Pansini G, Luque RM, Castaño JP, degli Uberti E, Zatelli MC (2015) The expression of the truncated isoform of somatostatin receptor subtype 5 associates with aggressiveness in medullary thyroid carcinoma cells. Endocrine 50:442-52. PubMed |
| Rossi M, Buratto M, Tagliati F, Rossi R, Lupo S, Trasforini G, Lanza G, Franceschetti P, Bruni S, Degli Uberti E, Zatelli MC (2015) Relevance of BRAF(V600E) mutation testing versus RAS point mutations and RET/PTC rearrangements evaluation in the diagnosis of thyroid cancer. Thyroid 25:221-8. PubMed |
| Zatelli MC, Gagliano T, Pelà M, Bianco S, Bertolasi V, Tagliati F, Guerrini R, degli Uberti E, Salvadori S, Trapella C (2014) N-carbamidoyl-4-((3-ethyl-2,4,4-trimethylcyclohexyl)methyl)benzamide enhances staurosporine cytotoxic effects likely inhibiting the protective action of Magmas toward cell apoptosis. J. Med. Chem. 57:4606-14. PubMed |
| Gagliano T, Filieri C, Minoia M, Buratto M, Tagliati F, Ambrosio MR, Lapparelli M, Zoli M, Frank G, degli Uberti E, Zatelli MC (2013) Cabergoline reduces cell viability in non functioning pituitary adenomas by inhibiting vascular endothelial growth factor secretion. Pituitary 16:91-100. PubMed |
| Gagliano T, Bellio M, Gentilin E, Molè D, Tagliati F, Schiavon M, Cavallesco NG, Andriolo LG, Ambrosio MR, Rea F, Degli Uberti E, Zatelli MC (2013) mTOR, p70S6K, AKT, and ERK1/2 levels predict sensitivity to mTOR and PI3K/mTOR inhibitors in human bronchial carcinoids. Endocr. Relat. Cancer 20:463-75. PubMed |
| Gentilin E, Tagliati F, Filieri C, Molè D, Minoia M, Rosaria Ambrosio M, Degli Uberti EC, Zatelli MC (2013) miR-26a plays an important role in cell cycle regulation in ACTH-secreting pituitary adenomas by modulating protein kinase Cδ. Endocrinology 154:1690-700. PubMed |
| Molè D, Gentilin E, Gagliano T, Tagliati F, Bondanelli M, Pelizzo MR, Rossi M, Filieri C, Pansini G, degli Uberti EC, Zatelli MC (2012) Protein kinase C: a putative new target for the control of human medullary thyroid carcinoma cell proliferation in vitro. Endocrinology 153:2088-98. PubMed |
| Minoia M, Gentilin E, Molè D, Rossi M, Filieri C, Tagliati F, Baroni A, Ambrosio MR, degli Uberti E, Zatelli MC (2012) Growth hormone receptor blockade inhibits growth hormone-induced chemoresistance by restoring cytotoxic-induced apoptosis in breast cancer cells independently of estrogen receptor expression. J. Clin. Endocrinol. Metab. 97:E907-16. PubMed |
| Rossi M, Buratto M, Bruni S, Filieri C, Tagliati F, Trasforini G, Rossi R, Beccati MD, Degli Uberti EC, Zatelli MC (2012) Role of ultrasonographic/clinical profile, cytology, and BRAF V600E mutation evaluation in thyroid nodule screening for malignancy: a prospective study. J. Clin. Endocrinol. Metab. 97:2354-61. PubMed |
| Molè D, Gagliano T, Gentilin E, Tagliati F, Pasquali C, Ambrosio MR, Pansini G, Degli Uberti EC, Zatelli MC (2011) Targeting protein kinase C by Enzastaurin restrains proliferation and secretion in human pancreatic endocrine tumors. Endocr. Relat. Cancer 18:439-50. PubMed |
| Zatelli MC, Minoia M, Martini C, Tagliati F, Ambrosio MR, Schiavon M, Buratto M, Calabrese F, Gentilin E, Cavallesco G, Berdondini L, Rea F, degli Uberti EC (2010) Everolimus as a new potential antiproliferative agent in aggressive human bronchial carcinoids. Endocr. Relat. Cancer 17:719-29. PubMed |
| Romei C, Mariotti S, Fugazzola L, Taccaliti A, Pacini F, Opocher G, Mian C, Castellano M, degli Uberti E, Ceccherini I, Cremonini N, Seregni E, Orlandi F, Ferolla P, Puxeddu E, Giorgino F, Colao A, Loli P, Bondi F, Cosci B, Bottici V, Cappai A, Pinna G, Persani L, Verga U, Uberta V, Boscaro M, Castagna MG, Cappelli C, Zatelli MC, Faggiano A, Francia G, Brandi ML, Falchetti A, Pinchera A, Elisei R, (2010) Multiple endocrine neoplasia type 2 syndromes (MEN 2): results from the ItaMEN network analysis on the prevalence of different genotypes and phenotypes. Eur. J. Endocrinol. 163:301-8. PubMed |
| Zatelli MC, Gentilin E, Daffara F, Tagliati F, Reimondo G, Carandina G, Ambrosio MR, Terzolo M, Degli Uberti EC (2010) Therapeutic concentrations of mitotane (o,p'-DDD) inhibit thyrotroph cell viability and TSH expression and secretion in a mouse cell line model. Endocrinology 151:2453-61. PubMed |
| Zatelli MC, degli Uberti E (2009) The significance of new somatostatin analogs as therapeutic agents. Curr Opin Investig Drugs 10:1025-31. PubMed |
The managers of the Service collaborate widely with other research groups both at home and abroad. Some examples of these are:
INTERNATIONAL PATENTS
“Methods of modulating the proliferation of medullary thyroid carcinoma cells” degli Uberti EC, Zatelli MC, Culler, MD. Applicant’s or agent’s file reference: 50165/006W02; Biomeasure Incorporated, Milford, MA, USA; International application number: PCT/US02/06729.
“Pharmaceutical compositions which inhibit proliferation of pituitary adenomas and method of use thereof” degli Uberti EC, Zatelli MC, Culler, MD. Applicant’s or agent’s file reference: 50165/010W02; Biomeasure Incorporated, Milford, MA, USA; International application number: PCT/US02/19998.
"Magmas inhibitors to sensitize tumor cells to therapy" Trapella C, Guerrini R, Salvadori S, Bianco S, Gagliano T, Tagliati F, Zatelli MC, degli Uberti E. International application number: 14230174, depositato il 31 marzo 2014.
NATIONAL PATENTS
"Uso di inibitori delle Cicloossigenasi di tipo 2 come agenti atti a modificare la farmacoresistenza cellulare" depositato il 1 Agosto 2005 con N.RM2005A000408.
We provide experimental design guidance and custom services. Customers will get a qualified quotation for each services as soon as possible for free.
For routine analysis prices can be arranged (subscription may be possible).
Needs assistance, or have a question, please contact us.
| Code no. | Brief description of the services we offer | Price (€) |
| IM-B-001 | DNA extraction | From blood: 2.00/sample From cell pellets: 2.00/sample From tissue: 4.00/sample |
| IM-B-002 | Nucleic acids extraction from paraffin-embedded samples | 7.00/slide |
| IM-B-003 | Complete RNA extraction with quality report | From blood:8.00/ sample From cell pellets: 8.00/sample From tissue: 12.00/ sample |
| IM-B-004 | Qualitative PCR | 5.00/reaction |
| IM-B-005 | Real-Time PCR | Upon request, according to necessary technology |
| IM-B-006 | Primer design | 10.00/copy |
| IM-B-007 | Probe design | Per TaqMan Real-Time PCR: 5.00/probe |
| IM-B-008 | Plate reading (Real Time PCR) | Without data processing: 5.00/plate |
| IM-B-009 | Including data processing: 10.00/plate | |
| IM-B-010 | Plate reading (fluorescence, luminescence, absorbance, AlphaScreen technology) |
Without data processing: 2.00/plate |
| IM-B-011 | Including data processing: 6.00/plate | |
| IM-B-012 | Expansion of endocrine and endocrine-related cell lines (standard medium) price per T75 flask/week | 20.00 |
| IM-B-013 | Primary culture of endocrine and endocrine-related tissue explants | 200.00 |
| IM-B-014 | Cell viability assay after drug test | cell line and drug supplied: 5.00/sample only drug supplied: 10.00/sample |
| IM-B-015 | Caspase 3/7 activity luminescence assay | 5.00/sample |
| IM-B-016 | Quantification of secreted hormone after drug test | Upon request |
| IM-B-017 | Alpha screen | 1000,00-3000,00 price varies according to the number of samples and number of signal transduction pathways to be analysed |
| IM-B-018 | Luciferase activity assays | Upon request |
| Mini-kit to test bioactive molecules on cell cultures | ||
| 1-Cell Viability assays on cell cultures | ||
| IM-B-019 | Cell culture expansion and evaluation of the effects of bioactive molecules by colorimetric or luminescent cell viability assay and/or caspase assay | TBA (To be agreed) |
| IM-B-020 | Preparation of primary cell cultures and evaluation of the effects of bioactive molecules by colorimetric or luminescent cell viability assay and/or caspase assay | TBA |
| 2-Cell function assays | ||
| IM-B-021 | Measurement of the levels of secreted bioactive substances in cell culture medium | TBA |
| IM-B-022 | Qualitative and quantitative analysis of gene expression in cell cultures in response to exogenous substances | TBA |
| IM-B-023 | Analysis of intracellular pathways in response to exogenous substances | TBA |
| 3-Genotype-phenotype correlation studies | ||
| IM-B-024 | DNA extraction and analysis of genetic markers in cell cultures or tissues | TBA |
Some of the services/products offered may be provided with the help of other services of the LTTA Laboratory.
Each service includes a description of the procedure and supply of images for scientific publication purposes.
Prices are shown without VAT and are subjected to change according to the cost of consumables necessary (every 12 months).
Our Service combines several decades of experience in the synthesis, purification and characterisation of organic, peptide and peptidomimetic molecules. In the course of our academic work we have identified many bioactive molecules capable of interacting with G protein-coupled receptors (GPCRs). In particular, compounds capable of interacting (as agonists, antagonists and partial agonists) with opioid receptors, the nociceptin receptor, the urotensin receptor and the neuropeptide S receptor. Various molecules identified by us here are now available commercially and used as reference standards in many academic and industrial laboratories working in the field of GPCRs.
The level of scientific work we do can be seen in many papers in prestigious international journals.
Recently, through a collaboration between the Neurology department of the hospital S.Anna di Ferrara and the Genzyme Corporation of Boston (USA), the efficacy of a recombinant enzyme (Myozyme) was demonstrated in the treatment of the rare metabolic condition, Pompe disease. Using mass spectrometry, the presence of marked glucose was evaluated in affected patients. As part of the same project a number of marked tetrasaccharides were identified in the urine of these patients.
HPLC (High Performance Liquid Chromatography) is an analytical tool derived from traditional chromatography and based on the same principles. In traditional chromatography the main component is the column that contains the stationary phase inside of which the mobile phase flows, represented by the eluent. The passage of the eluent occurs through the pressure exerted by the column of liquid forming the mobile phase and thus the process, if the stationary phase is not sufficiently porous, may also be very slow. Separation of the components occurs through the interactions that come about between the constituents of the mixture and the two phases. The interactions that occur may be electrostatic, dipole-dipole, van der Waals forces or else the result of an ionic exchange mechanism. This depends on the properties of both the two phases and the mixture to be separated. In HPLC the force that enables the eluent to flow down through the column is the pressure that is applied by a pump at the head of the column and that forces the mobile phase to flow inside the stationary phase. This not only makes the process quicker but also means that a greater number of theoretical plates can be obtained, which means a better resolution. The theoretical plate of a chromatography column corresponds to the section of a column where a chemical species is in equilibrium between the two phases (stationary and mobile) prior to the eluent drawing it into the next phase. However, the thinner the theoretical plate, the greater will be the efficiency of the column. The first positive developments in fluid chromatography were made after it was suggested and found that the theoretical plate could be made thinner by packing smaller particles into the column. The technology to make particles of sizes varying from 5 to 10 μm to pack into the column was not developed until the end of the 1960s. The name High Performance Liquid Chromatography (HPLC) serves to distinguish the new chromatography technique from traditional chromatography.
Mass spectrometry has been described as the smallest scale in the world, not because of the instrument’s size but because of the size of what it measures – molecular mass. Recent decades have seen considerable technological evolution in mass spectrometry, so that it is now possible to use it to determine peptides, proteins, nucleic acids, carbohydrates, drugs and other biologically relevant molecules.
Due to ionisation sources such as electrospray ionisation (ESI) and matrix-assisted laser desorption/ ionisation (MALDI), mass spectrometry has become an irreplaceable tool in biological science.
A mass spectrometer determines the mass, i.e. the molecular mass, of molecules by measuring the mass-to-charge ratio (m/z) of its ion. The ions necessary for this determination are generated by inducing either the loss or gain of a charge from neutral molecules. Once formed, the charged ions are directed into the mass analyser where they are separated according to their mass/charge ratio and finally detected. The result of molecular ionisation, ion separation and ion detection is a spectrum that can provide the molecular mass of the substance(s) present in the sample(s).
A mass spectrometer determines the mass of a molecule by measuring the mass-to-charge ratio (m/z) of its ion. Ions are generated by inducing either the loss or gain of a charge from a neutral species. Once formed, ions are electrostatically directed into a mass analyser where they are separated according to m/z and finally detected. The result of molecular ionization, ion separation, and ion detection is a spectrum that can provide molecular mass and even structural information.
Determination of accurate mass of an analyte
The service can provide the accurate mass of compounds, both in mixture or as single compound using the latest technology HPLC- Chip- MS-Q-TOF instrument. The Agilent ESI-Q-TOF 6520® is a mass spectrometer coupled with a Agilent nano HPLC Chip Cube® equipped with a double quadrupole and a TOF analyser that allows determination of the exact mass (error less than 2 ppm) of molecules between 200 to 20000 Daltons molecular weight. The possibility to separate complex mixture by nano-HPLC afford the analysis of metabolites or biological fluid to determine their exact mass and hopefully the structure of the molecule. The latest possibility is allowed by the quadrupole ion trap that provide the MS/MS analysis for the determination of the molecular structure or the peptide sequence.
Nano-HPLC-Chip-Cube® HRMS Agilent 6520 ESI-Q-TOF
Molecular Interactions, Biomarkers and Delivery facility
Laboratorio per le Tecnologie delle Terapie Avanzate (LTTA Centre)
Area Polo Chimico Biomedico "CUBO" - second floor
Via Fossato di Mortara 70 - 44121 Ferrara
+39 0532 455859 - 455856 - 455767
Prof. Remo Guerrini
remo.guerrini@unife.it
+39 0532 455988
Dr Claudio Trapella (Ph.D., AMRSC)
claudio.trapella@unife.it
+39 0532 455924
Dr Erika Marzola
Prof. Claudio Trapella
Dr Anna Talarico
anna.talarico@unife.it
Prof. Matteo Marti
matteo.marti@unife.it
Prof. Margherita Neri
margherita.neri@unife.it
| Ruzza C, Rizzi A, Malfacini D, Cerlesi MC, Ferrari F, Marzola E, Ambrosio C, Gro C, Severo S, Costa T, Calo G, Guerrini R (2014) Pharmacological characterization of tachykinin tetrabranched derivatives. Br. J. Pharmacol. 171:4125-37. PubMed |
| Pelà M, Saxena P, Luciani R, Santucci M, Ferrari S, Marverti G, Marraccini C, Martello A, Pirondi S, Genovese F, Salvadori S, D'Arca D, Ponterini G, Costi MP, Guerrini R (2014) Optimization of peptides that target human thymidylate synthase to inhibit ovarian cancer cell growth. J. Med. Chem. 57:1355-67. PubMed |
| Oishi M, Kushikata T, Niwa H, Yakoshi C, Ogasawara C, Calo G, Guerrini R, Hirota K (2014) Endogenous neuropeptide S tone influences sleep-wake rhythm in rats. Neurosci. Lett. 581:94-7. PubMed |
| Pelà M, Del Zoppo L, Allegri L, Marzola E, Ruzza C, Calo G, Perissutti E, Frecentese F, Salvadori S, Guerrini R (2014) Racemic synthesis and solid phase peptide synthesis application of the chimeric valine/leucine derivative 2-amino-3,3,4-trimethyl-pentanoic acid. Pharmazie 69:496-9. PubMed |
| Smilkov K, Janevik E, Guerrini R, Pasquali M, Boschi A, Uccelli L, Di Domenico G, Duatti A (2014) Preparation and first biological evaluation of novel Re-188/Tc-99m peptide conjugates with substance-P. Appl Radiat Isot 92:25-31. PubMed |
| Rizzi A, Malfacini D, Cerlesi MC, Ruzza C, Marzola E, Bird MF, Rowbotham DJ, Salvadori S, Guerrini R, Lambert DG, Calo G (2014) In vitro and in vivo pharmacological characterization of nociceptin/orphanin FQ tetrabranched derivatives. Br. J. Pharmacol. 171:4138-53. PubMed |
| Guerrini R, Marzola E, Trapella C, Pela' M, Molinari S, Cerlesi MC, Malfacini D, Rizzi A, Salvadori S, Calo' G (2014) A novel and facile synthesis of tetra branched derivatives of nociceptin/orphanin FQ. Bioorg. Med. Chem. 22:3703-12. PubMed |
| Zatelli MC, Gagliano T, Pelà M, Bianco S, Bertolasi V, Tagliati F, Guerrini R, degli Uberti E, Salvadori S, Trapella C (2014) N-carbamidoyl-4-((3-ethyl-2,4,4-trimethylcyclohexyl)methyl)benzamide enhances staurosporine cytotoxic effects likely inhibiting the protective action of Magmas toward cell apoptosis. J. Med. Chem. 57:4606-14. PubMed |
| Molinari S, Camarda V, Rizzi A, Marzola G, Salvadori S, Marzola E, Molinari P, McDonald J, Ko MC, Lambert DG, Calo' G, Guerrini R (2013) [Dmt1]N/OFQ(1-13)-NH2: a potent nociceptin/orphanin FQ and opioid receptor universal agonist. Br. J. Pharmacol. 168:151-62. PubMed |
| Thompson AA, Liu W, Chun E, Katritch V, Wu H, Vardy E, Huang XP, Trapella C, Guerrini R, Calo G, Roth BL, Cherezov V, Stevens RC (2012) Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic. Nature 485:395-9. PubMed |
Members of our Service have worked in many collaborations with other research groups both at home and abroad. Examples of some of these are:
University Depts. Cardiovascular Sciences, Div. Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester UK.
Université Pierre et Marie Curie - UPMC, UMR 7201 CNRS, Institut Parisien de Chimie Moléculaire IPCM, 4, Place Jussieu, Bâtiment F, 2ème étage, porte 217, boîte 183, 75252, Paris, Cedex 05, France.
Department of Organic and Medicinal Chemistry University of Granada (Spain) Prof.ssa Maria del Carmen Nunez Carretero.
Dipartimento di Chimica, Università degli Studi di Napoli "Federico II", Complesso Universitario di Monte S. Angelo, via Cintia, 80126 Napoli, Italy.
National Institute for Medical Research, Medical research Cuncil, The ridgeway, Mill Hill, London NW7 1AA, UK.
INTERNATIONAL PATENTS
“Analogs of Nociceptin” Guerrini R, Calo’ G, Salvadori S, Regoli D. Application number: EP 1422240.
“Highly potent full and partial agonists and antagonists of the nociceptin/orphanin FQ receptor” Guerrini R, Calo' G, Regoli D, Salvadori S. Application number: PCT/EP2006/050958; WO2006087340.
“Biologically potent analogues of the Dmt-Tic pharmacophore and methods of use” Lazarus L H, Salvadori S, Balboni G, Guerrini R. Application number: US 2006-0104907.
“Magmas Inhibitors to sensitize tumor cells to therapy” Zatelli MC, Tagliati F, degli Uberti E, Gagliano T. Salvadori S. Guerrini R. Bianco S. Trapella C. Application number: US- EFS ID: 18623727-2014.
| Code no. | Brief description of the services we offer | Price (€ per hour) |
| Preliminary consultation on formulation of experimental design; during the consultation an estimate of the overall cost of the analyses can be provided. The Service offers consultations for specific projects, and estimates of the overall cost are provided free of charge according to the needs of the client. Please contact us for special requirements not shown in the price list, since the packages offered can be tailored to requirements outside the standard service. For routine analysis prices can be arranged (subscriptions may be possible). |
0.00 | |
| REVERSED PHASE | ||
| LTTA-IM-CHSM-02 | Method development | 800.00 |
| LTTA-IM-CHSM-02 | Cost for each analysis | 120.00 |
Some of the services/products offered may be provided with the help of other services of the LTTA Laboratory.
Each service includes a description of the procedure and supply of images for scientific publication purposes.
Prices are shown without VAT and are subjected to change according to the cost of consumables necessary (every 12 months).
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