PROTEOMIC APPROACHES IN DRUGS AND BIOMARKERS DISCOVERY

DOTTORATO DI RICERCA IN CHIMICA DEL FARMACO (XXV CICLO) Proteomic approaches in Drugs and Biomarkers Discovery Dott. Andrea Pancotti Tutor: Prof. Marina Carini Co-tutor: Prof. Sergio Romeo Abstract My thesis project is focused on development and application of proteomic approaches in two fundamental research areas: the drug discovery process and the discovery of disease biomarkers. In particular, new and reliable strategies by mass spectrometry for the fast identification and validation of drug targets, lead compounds and disease biomarkers have been considered. Proteomic Approaches in Drug Discovery In Prof. Romeo\u2019s group new compounds have been identified with high in vitro efficacy against the intraerythrocytic stages of Plasmodium falciparum (P.f.) (IC50 <10 nM),1 but the molecular target is unknown and preliminary results indicate that these compounds may exerts their activity against P.f. through multiple targets. Proteomics offers a unique tool for target identification2 and several proteomic approaches are available, one of the most interesting is the so called chemical proteomics, which couple affinity purification methods with mass spectrometry and therefore permits to increase selectivity and sensitivity.3 In order to facilitate the mass spectrometric analysis, it would be necessary to know the target localization into P.f. of our compounds. Therefore last year, fluorescence compounds, as analytical tools for preliminary target identification, were designed. These compounds have been tested in the Prof. Taramelli\u2019s laboratory against D10 and W2 strains, but only few compounds resulted to be active, and activities were elicited only in a micromolar range. These activities were not sufficient to conduct fluorescent studies, therefore further optimizations were undertaken. Considering the recent SAR obtained in our laboratory, compound 1 has been synthesized characterized by the presence of coumarin and a propylendiamine linker between leucine and the fluorescent group. This compound was active against P.f (IC50 D10 and W2= 13 nM) and fluorescent studies are being performed. In the meanwhile we are focusing our attention on the target identification. The adopted chemical proteomics approach requires the preparation of an alkyne derivate of the lead compound that will be incubated in a Pf lysate. Then trough click chemistry the protein bound alkyne derivative will be reacted with an azide containing a cleavable linker and a terminal biotin unit (compound 3). The lysate will be purified by affinity based chromatography using streptavidin beads. Then the cleavable linker will be cleaved and the protein-compound complex analyzed using HPLC-MS analysis. Thus propargylic derivative 2 was synthesized. It was tested in the Professor Taramelli\u2019s laboratory and resulted to be very active against Pf. (IC50 D10= 8nM/ IC50 W2= 12nM). Compound 3 has been synthesized and it is characterized by a biotin unit, an acylhydrazone cleavable linker, a poliethylenglicol spacer and a terminal azide. In order to verify if compound 2 and 3 were useful tools to perform the procedure that we aimed to follow, the optimized click chemistry reaction was performed between the two compounds obtaining compound 4. In order to test the whole proteomics procedure, compound 4 was immobilized on agarose streptavidin beads and after the selective cleavage of the acylhydrazone cleavable linker and reduction of intermediate, compound 5 was obtained and characterized through direct infusion ESI-MS spectrum. After this successful result the final proteomics analysis is being conducted in collaboration with Professor Taramelli\u2019s laboratory. Proteomic Approaches in Biomarkers Discovery One of the most exciting areas of proteomic research is the identification and validation of disease biomarkers which can be used as measurements within clinical studies and for the purpose of predictive diagnosis. The study of proteomics is considered to be a key for the characterization of human diseases and disease states, and mass spectrometry technology plays a crucial role in this disease research. We will focus in particular on detection and quantization of post-translational modifications of proteins caused by oxidative and carbonyl stress. While the development of specific antibodies against modified proteins has made it possible to confirm the occurrence of oxidative stress in vivo and its involvement in several physio-pathological conditions, the resultant chemical modifications of proteins has not yet been explored. Hence we need proteomic tools, which can sensitively detect oxidative damage on peptides and proteins: these tools would be helpful to understand exactly when, how, and where the damage occurs, and to gain a deeper insights into the mechanism of onset, progression, and/or complication of the diseases. Proteomics approaches have led to the identification of the protein/s showing high sensitivity to oxidation/carbonylation, and among them human serum albumin (HSA) was found to be the main target in the circulation.4-6 During the first year we demonstrated covalent modification of HSA-Cys34 by acrolein in patients undergoing hepatectomy surgery by using a mass spectrometric approach based on a triple quadruple mass spectrometer in precursor ion scan mode, able to identify unknown modifications of Cys34 by reactive carbonyl species (RCS) generated by lipid peroxidation. However, parallel studies performed in our laboratories demonstrated that in some pathologic conditions (i.e. uremic subjects), Cys34 undergoes disulfide formation by cysteinylation, thus limiting its availability for reaction with RCS. Hence, Cys 34-adducts couldn\u2019t always be useful as carbonylation biomarkers. Therefore, I focused my attention on another HSA nucleophilic site (His-146), previously recognized as a potential oxidation/carbonylation target7. Digesting HSA with tripsin or tripsin+chimotripsin, two different peptides containing His-146 have been obtained: H*PYFYAPELLFFAK and H*PY, respectively. Anyway, analysis of tripsin or tripsin+chimotripsin digested HNE-HSA adduct did not lead to identification of any covalent modifications on His 146. Using an high-resolution, high mass accuracy mass spectrometer (LTQ-Orbitrap), performing a full scan analysis in data-dependent mode followed by data analysis with the SEQUEST algorithm it was possible to explain that the absence of any signals relative to RCS-H*PYFYAPELLFFAK adducts was due to different missed cleavages. The analyses relative to the H*PY peptide indicated the formation of one adduct only, H(HNE)PYF, but no significant fragmentation of the corresponding [M+H]+ was observed in MS/MS experiments, even working at high collision energy (40eV). Maybe the reason could be that the peptide is too short for an optimal fragmentation. Taking into account these observations, we have considered as alternative approach the application of a new HSA digestion strategy, based on the use of chimotripsin only. In silico studies indicated the EIARRH*PY peptide as a new tag containing the target residue His 146 in the middle, exactly like Cys-34 in the LQQC*PF peptide, previously analyzed with satisfactory results. This new peptide has also the appropriate length to be analyzed by HPLC-MS/MS. Hence, the EIARRH*PY peptide seemed to possess the optimal characteristics to start a new MS strategy involving His-146 as a carbonyl adduction site on HSA. Thus, HSA was isolated from human plasma by affinity chromatography, reduced with NaBH4 and digested with chimotripsin only in order to obtain the expected peptide. The peptide mixture was analyzed by LTQ-Orbitrap in data dependent scan mode and afterwards by the SEQUEST algorithm, comparing the obtained data with the primary sequence of the protein. The results of the analysis confirmed the formation of the expected peptide. In this way we created the bases for the future studies on the EIARRH*PY peptide, in order to identify any covalent modifications of His146 induced by RCS. References 1. Vaiana, N.; Marzahn, M.; Parapini, S.; Liu, P.; DelAgli, M.; Pancotti, A.; Sangiovanni, E.; Basilico, N.; Bosisio, E.; Dunn, B. M.; Taramelli, D.; Romeo, S. Bioorganic & Medicinal Chemistry Letters 2012, 22, 5915. 2. Brown, D.; Superti-Furga, G. Drug Discov Today 2003, 8, 1067. 3. Godl, K.; Wissing, J.; Kurtenbach, A.; Habenberger, P.; Blencke, S.; Gutbrod, H.; Salassidis, K.; Stein-gerlach, M.; Missio, A.; Cotten, M.; Daub, H. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 15434. 4. Mera, K.; Anraku, M.; Kitamura, K.; Nakajou, K.; Maruyama, T.; Otagiri, M. Biochem. Biophys. Res. Commun. 2005, 334, 1322. 5. Anraku, M.; Kitamura, K.; Shinohara, A.; Adachi, M.; Suenaga, A.; Maruyama, T.; Miyanaka, K.; Miyoshi, T.; Shiraishi, N.; Nonoguchi, H.; Otagiri, M.; Tomita, K. Kidney Int. 2004, 66, 841. 6. Aldini, G.; Vistoli, G.; Regazzoni, L.; Gamberoni, L.; Facino, R. M.; Yamaguchi, S.; Uchida, K.; Carini, M. Chem. Res. Toxicol. 2008, 21, 824. 7. Aldini, G.; Gamberoni, L.; Orioli, M.; Beretta, G.; Regazzoni, L.; Facino, R. M.; Carini, M. J. Mass Spectrom. 2006, 41, 1149

Multiple scattering x-ray photoelectron diffraction study of the SrTiO3(100) surface

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)The atomic surface structure of SrTiO3(100) after annealing at 630 degrees C in vacuum is investigated by x-ray photoelectron diffraction (XPD) using the Sr 3d(5/2) core level. The photoelectron diffraction peaks are successfully assigned by considering the forward scattering of photoelectrons by the atomic potential near the emitter atom in the lattice. The strongest diffraction peaks are aligned along the single crystal internuclear axes. We compare the results of photoelectron multiple scattering calculations (MSC) of SrO and TiO2 terminated SrTiO3(100) surfaces, including surface relaxation and rumpling, with the experimental data. For TiO2 and SrO terminated SrTiO3(100) surfaces, all top-layer cations relax inward, whereas second-layer atoms relax outward. The surface rumpling for SrO- and TiO2-terminated surfaces agrees well with low-energy electron diffraction results. Using a genetic algorithm the best agreement of MSC to the experimental XPD data is obtained for a SrO terminated surface with a 30% coverage of 3 ML SrO(100) islands.1063Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)European Commission [NMP3-CT-2005-013862]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [0554993-4]European Commission [NMP3-CT-2005-013862

Multiple Scattering X-ray Photoelectron Diffraction Study Of The Srtio 3 (100) Surface

The atomic surface structure of SrTiO3 (100) after annealing at 630 °C in vacuum is investigated by x-ray photoelectron diffraction (XPD) using the Sr 3 d5/2 core level. The photoelectron diffraction peaks are successfully assigned by considering the forward scattering of photoelectrons by the atomic potential near the emitter atom in the lattice. The strongest diffraction peaks are aligned along the single crystal internuclear axes. We compare the results of photoelectron multiple scattering calculations (MSC) of SrO and TiO2 terminated SrTiO3 (100) surfaces, including surface relaxation and rumpling, with the experimental data. For TiO2 and SrO terminated SrTiO3 (100) surfaces, all top-layer cations relax inward, whereas second-layer atoms relax outward. The surface rumpling for SrO- and TiO2 -terminated surfaces agrees well with low-energy electron diffraction results. Using a genetic algorithm the best agreement of MSC to the experimental XPD data is obtained for a SrO terminated surface with a 30% coverage of 3 ML SrO(100) islands. © 2009 American Institute of Physics.1063Rijnders, G., Curras, S., Huijben, M., Blanck, D., Rogalla, H., Kotecki, D.E., Baniecki, J.D., Wise, R., (2004) Appl. Phys. Lett., 84, p. 1150. , 0003-6951, () 10.1063/1.1646463;, IBM J. Res. Dev. 0018-8646 43, 367 (1999)Kawasaki, M., Ohtomo, A., Arakane, T., Takahashi, K., Yoshimoto, M., Koinuma, H., Atomic control of SrTiO3 surface for perfect epitaxy of perovskite oxides (1996) Applied Surface Science, 107, pp. 102-106. , DOI 10.1016/S0169-4332(96)00512-0, PII S0169433296005120Erdman, N., Marks, L.D., (2003) Surf. Sci., 526, p. 107. , 0039-6028,. 10.1016/S0039-6028(02)02573-6Kubo, T., Nozoye, H., (2001) Phys. Rev. Lett., 86, p. 1801. , 0031-9007,. 10.1103/PhysRevLett.86.1801Szot, K., Speier, W., Breuer, U., Meyer, R., Szade, J., Waser, R., (2000) Surf. Sci., 460, p. 112. , 0039-6028,. 10.1016/S0039-6028(00)00522-7Gunhold, A., Gomann, K., Beuermann, L., Frerichs, M., Borchardt, G., Kempter, V., Maus-Friedrichs, W., Geometric structure and chemical composition of SrTiO3 surfaces heated under oxidizing and reducing conditions (2002) Surface Science, 507-510, pp. 447-452. , DOI 10.1016/S0039-6028(02)01284-0, PII S0039602802012840Szot, K., Speier, W., (1999) Phys. Rev. B, 60, p. 5909. , 0163-1829,. 10.1103/PhysRevB.60.5909Castell, M.R., Nanostructures on the SrTiO3(0 0 1) surface studied by STM (2002) Surface Science, 516 (1-2), pp. 33-42. , DOI 10.1016/S0039-6028(02)02053-8, PII S0039602802020538Ruddlesden, S.N., Popper, P., (1958) Acta Crystallogr., 11, p. 54. , 0907-4449,. 10.1107/S0365110X58000128Liang Yong, Bonnell Dawn, A., Atomic structures of reduced SrTiO3(001) surfaces (1993) Surface Science, 285 (3), pp. L510-L516. , DOI 10.1016/0039-6028(93)90422-GHenrich, V.E., Dresselhaus, G., Zeiger, H.J., (1978) Phys. Rev. B, 17, p. 4908. , 0163-1829,. 10.1103/PhysRevB.17.4908Matsumoto Takuya, Tanaka Hiroyuki, Kawai Tomoji, Kawai Shichio, STM-imaging of a SrTiO3(100) surface with atomic-scale resolution (1992) Surface Science, 278 (3), pp. L153-L158. , DOI 10.1016/0039-6028(92)90659-THaruyama, Y., Fukutani, H., Aiura, Y., Nishihara, Y., Komeda, T., Kodaira, S., Maryama, T., Kato, H., (1993) Jpn. J. Appl. Phys., Suppl., 32, p. 543. , 10.1143/JJAP.32.L543Hirata, A., Saika, K., Koma, A., Ando, A., (1994) Surf. Sci., 319, p. 267. , 0039-6028,. 10.1016/0039-6028(94)90593-2Zagonel, L.F., Barrett, N., Renault, O., Bailly, A., Bäurer, M., Hoffmann, M., Shih, S.-J., Cockayne, D., (2008) Surf. Interface Anal., 40, p. 1709. , 0142-2421,. 10.1002/sia.2886Szot, K., Speier, W., Herion, J., Freiburg, C., (1997) Appl. Phys. A: Mater. Sci. Process., 64, p. 64. , 0947-8396Fompeyrine, J., Berger, R., Lang, H.P., Perret, J., Machler, E., Gerber, Ch., Locquet, J.-P., Local determination of the stacking sequence of layered materials (1998) Applied Physics Letters, 72 (14), pp. 1697-1699. , DOI 10.1063/1.121155, PII S0003695198019147Padilla, J., Vanderbilt, D., (1998) Surf. Sci., 418, p. 64. , 0039-6028,. 10.1016/S0039-6028(98)00670-0Cheng, C., Kunc, K., Lee, M.H., Structural relaxation and longitudinal dipole moment of SrTiO 3(001)(1 × 1) surfaces (2000) Physical Review B - Condensed Matter and Materials Physics, 62 (15), pp. 10409-10418. , DOI 10.1103/PhysRevB.62.10409Heifets, E., Kotomin, E.A., Maier, J., Semi-empirical simulations of surface relaxation for perovskite titanates (2000) Surface Science, 462 (1), pp. 19-35. , DOI 10.1016/S0039-6028(00)00603-8Bickel, N., Schmidt, G., Heinz, K., Muller, K., (1989) Phys. Rev. Lett., 62, p. 2009. , 0031-9007,. 10.1103/PhysRevLett.62.2009Hikita Tokihisa, Hanada Takashi, Kudo Masahiro, Kawai Maki, Structure and electronic state of the TiO2 and SrO terminated SrTiO3(100) surfaces (1993) Surface Science, 287 (88 PART A), pp. 377-381. , DOI 10.1016/0039-6028(93)90806-UIkeda, A., Nishimura, T., Morishita, T., Kido, Y., Surface relaxation and rumpling of TiO2-terminated SrTiO 3(001) determined by medium energy ion scattering (1999) Surface Science, 433, pp. 520-524. , DOI 10.1016/S0039-6028(99)00050-3Charlton, G., Brennan, S., Muryn, C.A., McGrath, R., Norman, D., Turner, T.S., Thornton, G., Surface relaxation of SrTiO3(001) (2000) Surface Science, 457 (1), pp. L376-L380. , DOI 10.1016/S0039-6028(00)00403-9Bottin, F., Finocchi, F., Noguera, C., (2003) Phys. Rev. B, 68, p. 035418. , 0163-1829,. 10.1103/PhysRevB.68.035418Eglitis, R.I., Vanderbilt, D., (2008) Phys. Rev. B, 77, p. 195408. , 0163-1829,. 10.1103/PhysRevB.77.195408Herger, R., Willmott, P.R., Bunk, O., Schlepütz, C.M., Patterson, B.D., Delley, B., Shneerson, V.L., Saldin, D.K., (2007) Phys. Rev. B, 76, p. 195435. , 0163-1829,. 10.1103/PhysRevB.76.195435Chen, Y., Hove, M.V., (1998), http://www.ap.cityu.edu.hk/personal-website/Van-Hove_files/mscd/mscdpack. html, MSCD Package, Lawrence Berkeley National LaboratoryPasierb, P., Komornicki, S., Rekas, M., Comparison of the chemical diffusion of undoped and Nb-doped SrTiO 3 (1999) Journal of Physics and Chemistry of Solids, 60 (11), pp. 1835-1844. , DOI 10.1016/S0022-3697(99)00193-6De Siervo, A., Soares, E.A., Landers, R., Kleiman, G.G., Photoelectron diffraction studies of Cu on Pd(111) random surface alloys (2005) Physical Review B - Condensed Matter and Materials Physics, 71 (11), pp. 1-7. , http://scitation.aip.org/getpdf/servlet/GetPDFServlet?filetype= pdf&id=PRBMDO000071000011115417000001&idtype=cvips, DOI 10.1103/PhysRevB.71.115417, 115417Mitchell, R.H., Chakhmouradian, A.R., Woodward, P.M., Crystal chemistry of perovskite-type compounds in the tausonite-loparite series, (Sr(1-2x)Na(x)La(x))TiO3 (2000) Physics and Chemistry of Minerals, 27 (8), pp. 583-589. , DOI 10.1007/s002690000103http://www.qivx.com, From the Integral Scientist Periodic Table of the Elements (ISPT), available onPendry, J., (1980) J. Phys C, 13, p. 937. , 10.1088/0022-3719/13/5/024 0081-1947Bondino, F., Comelli, G., Baraldi, A., Rosei, R., Lizzit, S., Goldoni, A., Larciprete, R., Paolucci, G., (2002) Phys. Rev. B, 66, p. 075402. , 0163-1829,. 10.1103/PhysRevB.66.075402Van Hove, M., Moritz, W., Over, H., Rous, P., Wander, A., Barbieri, A., Materer, N., Somorjai, G., (1993) Surf. Sci. Rep., 19, p. 191. , 0167-5729,. 10.1016/0167-5729(93)90011-DViana, M.L., Muio, R.D., Soares, E.A., Van Hove, M.A., De Carvalho, V.E., Global search in photoelectron diffraction structure determination using genetic algorithms (2007) Journal of Physics Condensed Matter, 19 (44), p. 446002. , DOI 10.1088/0953-8984/19/44/446002, PII S095389840753354

Foraminiferal biodiversity associated with cold-water coral carbonate mounds and open slope of SE Rockall Bank (Irish continental margin-NE Atlantic)

Cold-water coral (CWC) ecosystems are hotspots of macro- and microfaunal biodiversity and provide refuge for a wide variety of deep-sea species. We investigated how the abundance and biodiversity of 'live' (Rose Bengal stained) foraminifera varies with, and is related to, the occurrence of CWC on the Rockall Bank (NE Atlantic). Qualitative and quantitative analyses were performed on 21 replicate samples from 8 deep-sea stations, including 4 stations on CWC-covered carbonate mounds at depths of 567-657 m, and 4 stations on the adjacent slope at depths of 469-1958 m where CWC were absent. This sampling strategy enabled us to demonstrate that sediments surrounding the living CWC were characterised by higher foraminiferal abundance and biodiversity than open-slope sediments from the same area. A total of 163 foraminiferal species was identified. The dominant species in CWC sediments were: Spirillina vivipara, Allogromiid sp. 1. Globocassidulina subglobosa, Adercotryma wrighti, Eponides pusillus, Ehrenbergina carinata, Planulina ariminensis, Trochammina inflate and Paratrochammina challenged. Foraminifera were nearly absent in adjacent open slope areas subject to strong tidal currents and characterised by coarse grained deposits. We suggest that CWC create a heterogeneous three-dimensional substrate offering microhabitats to a diverse benthic foraminiferal community

Hafnium silicide formation on Si(100) upon annealing

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORHigh dielectric constant materials, such as HfO2, have been extensively studied as alternatives to SiO2 in new generations of Si based devices. Hf silicate/silicide formation has been reported in almost all literature studies of Hf based oxides on Si, using different methods of preparation. A silicate interface resembles close to the traditional Si/SiO2. The silicate very likely forms a very sharp interface between the Si substrate and the metal oxide, and would be suitable for device applications. However, the thermal instability of the interfacial silicate/oxide film leads to silicidation, causing a dramatic loss of the gate oxide integrity. Despite the importance of the Hf silicide surface and interface with Si, only a few studies of this surface are present in the literature, and a structural determination of the surface has not been reported. This paper reports a study of the Hf silicide formation upon annealing by using a combination of XPS, LEED, and x-ray photoelectron diffraction (XPD) analyses. Our results clearly indicate the formation of a unique ordered Hf silicide phase (HfSi2), which starts to crystallize when the annealing temperature is higher than 550 °C.High dielectric constant materials, such as HfO2, have been extensively studied as alternatives to SiO2 in new generations of Si based devices. Hf silicate/silicide formation has been reported in almost all literature studies of Hf based oxides on Si, using different methods of preparation. A silicate interface resembles close to the traditional Si/SiO2. The silicate very likely forms a very sharp interface between the Si substrate and the metal oxide, and would be suitable for device applications. However, the thermal instability of the interfacial silicate/oxide film leads to silicidation, causing a dramatic loss of the gate oxide integrity. Despite the importance of the Hf silicide surface and interface with Si, only a few studies of this surface are present in the literature, and a structural determination of the surface has not been reported. This paper reports a study of the Hf silicide formation upon annealing by using a combination of XPS, LEED, and x-ray photoelectron diffraction (XPD) analyses. Our results clearly indicate the formation of a unique ordered Hf silicide phase (HfSi2), which starts to crystallize when the annealing temperature is higher than 550 °C.747110FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORSem informaçãoSem informação170/04This work was financially supported by DAAD (PROBRAL D/03/23553) from Germany, and FAPESP, CNPq, and CAPES (PROBRAL 170/04) from Brazil. A.S. especially would like to thank CAPES for their support

A phase II randomised trial of 5-fluorouracil with or without interferon alpha-2a in advanced colorectal cancer.

With the association of 5-fluorouracil (5-FU) and alpha-interferon (IFN), objective responses as high as 26 63% have been reported in untreated patients with advanced colorectal cancer. However, grade 3-4 toxicity has also been reported. We have conducted a prospective phase II randomised study comparing 5-FU to 5-FU + IFN, to investigate whether the addition of IFN to a weekly 5-FU regimen devoid of significant toxicity used at our institutions could improve the effectiveness of 5-FU while maintaining acceptable toxicity. Patients with histologically proven advanced colorectal carcinoma were randomised to receive 5-FU 500 mg m-2 intravenous (i.v.) bolus on days 1-5 followed by 5-FU 500 mg m-2 i.v. bolus weekly from day 15, with or without IFN alpha-2a intramuscularly (i.m.) 1.5 mU daily on days 6-12 and 3 mU i.m. daily thereafter. The treatment was administered on an outpatient basis. Response was evaluated every 3 months, and treatment continued until progression or after two consecutive judgements of stable disease. Response rate was the main end point of the study. Of 141 patients eligible, 72 were randomised to 5-FU alone (arm A) and 69 to 5-FU + IFN (arm B). Responses were 9/72 (12.5%) in arm A and 6/69 (8.7%) in arm B; complete responses were three in arm A and two in arm B. Progression-free survival (median 4 months) and survival (median 12 months) were identical in the two arms. Toxicity was almost absent in arm A and moderate in arm B, represented mainly by haematological toxicity (usually leucopenia). In conclusion, overall survival was good in both arms of treatment and toxicity was moderate. While the response rate with 5-FU alone was in accord with the literature data, response to 5-FU + IFN was lower than expected. At least at this dosage and schedule, the association of 5-FU and IFN is no better than 5-FU alone and is of no clinical interest

Ferroelectricity in a quasiamorphous ultrathin BaTiO3 film

Until now, the quasiamorphous (QA) phase in BaTiO3 (BTO), SrTiO3 (STO), and BaZrO3 was achieved by pulling a thick film through a steep temperature gradient. Here, we show that a room-temperature deposited ultrathin film, subsequently annealed in O-2 can also produce a QA phase. The atomic, electronic, and ferroelectric (FE) structure of a QA, ultrathin BTO grown on STO were studied by x-ray diffraction (XRD), x-ray photoelectron diffraction (XPD), x-ray photoelectron spectroscopy (XPS), and piezoforce microscopy (PFM). The absence of long-range order is confirmed by in-and out-of-plane XRD as well as Ti 2p XPD. FE polarized domains with good retention have been successfully written into the QA film and exhibit a clear P-E hysteresis loop. Substrate clamping frustrates volume expansion during annealing leading to a QA film. Photoelectron spectroscopy confirms a similar overall electronic structure as for thicker films but with some significant differences. Simple charge-transfer arguments are not sufficient to explain the high-resolution core-level spectra. Ba, Ti, and O all show components associated with a surface region. We suggest that the observation of such a component in the Ti 2p spectrum is linked with the high dynamic charge tensor induced by the large off-center displacement of the Ti ion.8420French National Research Agency (ANR) [ANR-10-BLAN-1012]CEAFrench National Research Agency (ANR) [ANR-10-BLAN-1012

Discovery of oxybisbenzoylamides as a new class of antimalarial agents

We have previously described several potent dual inhibitors of Plasmodium falciparum (Pf) growth characterized by the presence of statin, a \u3b2-hydroxyl amino acid able to inhibit parasite\u2019s plasmepsins (PLM). While investigating the mechanism of action of these inhibitors new compounds deprived of statin were synthetized which lost the ability to inhibit PLM, but retained a significant Pf growth inhibition. Further structural simplifications showed that the inhibition of Pf viability was to ascribe to a new pharmacophore never described before as antimalaria

Breast cancer "tailored follow-up" in Italian oncology units: a web-based survey

urpose: Breast cancer follow-up procedures after primary treatment are still a controversial issue. Aim of this study was to investigate, through a web-based survey, surveillance methodologies selected by Italian oncologists in everyday clinical practice. Methods: Referents of Italian medical oncology units were invited to participate to the study via e-mail through the SurveyMonkey website. Participants were asked how, in their institution, exams of disease staging and follow-up are planned in asymptomatic women and if surveillance continues beyond the 5th year. Results: Between February and May 2013, 125 out of 233 (53.6%) invited referents of Italian medical oncology units agreed to participate in the survey. Ninety-seven (77.6%) referents state that modalities of breast cancer follow-up are planned according to the risk of disease progression at diagnosis and only 12 (9.6%) oncology units apply the minimal follow-up procedures according to international guidelines. Minimal follow-up is never applied in high risk asymptomatic women. Ninety-eight (78.4%) oncology units continue follow-up in all patients beyond 5 years. Conclusions: Our survey shows that 90.4% of participating Italian oncology units declare they do not apply the minimal breast cancer follow-up procedures after primary treatment in asymptomatic women, as suggested by national and international guidelines. Interestingly, about 80.0% of interviewed referents performs the so called "tailored follow-up", high intensity for high risk, low intensity for low risk patients. There is an urgent need of randomized clinical trials able to determine the effectiveness of risk-based follow-up modalities, their ideal frequency and persistence in time