Dosimetric comparison between VMAT with different dose calculation algorithms and protons for soft-tissue sarcoma radiotherapy.

BACKGROUND: To appraise the potential of volumetric modulated arc therapy (VMAT, RapidArc) and proton beams to simultaneously achieve target coverage and enhanced sparing of bone tissue in the treatment of soft-tissue sarcoma with adequate target coverage. MATERIAL AND METHODS: Ten patients presenting with soft-tissue sarcoma of the leg were collected for the study. Dose was prescribed to 66.5 Gy in 25 fractions to the planning target volume (PTV) while significant maximum dose to the bone was constrained to 50 Gy. Plans were optimised according to the RapidArc technique with 6 MV photon beams or for intensity modulated protons. RapidArc photon plans were computed with: 1) AAA; 2) Acuros XB as dose to medium; and 3) Acuros XB as dose to water. RESULTS: All plans acceptably met the criteria of target coverage (V95% >90-95%) and bone sparing (D(1 cm3) <50 Gy). Significantly higher PTV dose homogeneity was found for proton plans. Near-to-maximum dose to bone was similar for RapidArc and protons, while volume receiving medium/low dose levels was minimised with protons. Similar results were obtained for the remaining normal tissue. Dose distributions calculated with the dose to water option resulted ~5% higher than corresponding ones computed as dose to medium. CONCLUSION: High plan quality was demonstrated for both VMAT and proton techniques when applied to soft-tissue sarcoma.

Stereotactic body radiation therapy for liver tumours using flattening filter free beam: dosimetric and technical considerations.

PURPOSE: To report the initial institute experience in terms of dosimetric and technical aspects in stereotactic body radiation therapy (SBRT) delivered using flattening filter free (FFF) beam in patients with liver lesions. METHODS AND MATERIALS: From October 2010 to September 2011, 55 consecutive patients with 73 primary or metastatic hepatic lesions were treated with SBRT on TrueBeam using FFF beam and RapidArc technique. Clinical target volume (CTV) was defined on multi-phase CT scans, PET/CT, MRI, and 4D-CT. Dose prescription was 75 Gy in 3 fractions to planning target volume (PTV). Constraints for organs at risk were: 700 cc of liver free from the 15 Gy isodose, Dmax < 21 Gy for stomach and duodenum, Dmax < 30 Gy for heart, D0.1 cc < 18 Gy for spinal cord, V15 Gy < 35% for kidneys. The dose was downscaled in cases of not full achievement of dose constraints. Daily cone beam CT (CBCT) was performed. RESULTS: Forty-three patients with a single lesion, nine with two lesions and three with three lesions were treated with this protocol. Target and organs at risk objectives were met for all patients. Mean delivery time was 2.8 ± 1.0 min. Pre-treatment plan verification resulted in a Gamma Agreement Index of 98.6 ± 0.8%. Mean on-line co-registration shift of the daily CBCT to the simulation CT were: -0.08, 0.05 and -0.02 cm with standard deviations of 0.33, 0.39 and 0.55 cm in, vertical, longitudinal and lateral directions respectively. CONCLUSIONS: SBRT for liver targets delivered by means of FFF resulted to be feasible with short beam on time.

Can volumetric modulated arc therapy with flattening filter free beams play a role in stereotactic body radiotherapy for liver lesions? A volume-based analysis.

PURPOSE: To compare volumetric modulated arc therapy with flattening filter free (FFF) and flattening filter (FF) beams in patients with hepatic metastases subject to hypofractionated radiotherapy (RT). METHODS: A planning study on 13 virtual lesions of increasing volume was performed. Two single arc plans were optimized with the RapidArc technique using either FFF or FF beams. A second planning study was performed on ten patients treated for liver metastases to validate conclusions. In all cases, a dose of 75 Gy in 3 fractions was prescribed to the planning target volume (PTV) and plans were evaluated in terms of coverage, homogeneity, conformity, mean dose to healthy liver and to healthy tissue. For each parameter, results were expressed in relative terms as the percentage ratio between FFF and FF data. RESULTS: In terms of PTV coverage, conformity index favored FFF for targets of intermediate size while FF resulted more suitable for small (<100 cm(3)) and large (>300 cm(3)) targets. Plans optimized with FFF beams resulted in increased sparing of healthy tissue in ≈85% of cases. Despite the qualitative results, no statistically significant differences were found between FFF and FF results. Plans optimized with un-flattened beams resulted in higher average MU∕Gy than plans with FF beams. A remarkable and significant difference was observed in the beam-on time (BOT) needed to deliver plans. The BOT for FF plans was 8.2 ± 1.0 min; for FFF plans BOT was 2.2 ± 0.2 min. CONCLUSIONS: RapidArc plans optimized using FFF were dosimetrically equivalent to those optimized using FF beams, showing the feasibility of SBRT treatments with FFF beams. Some improvement in healthy tissue sparing was observed when using the FFF modality due to the different beam's profile. The main advantage was a considerable reduction of beam-on time, relevant for SBRT techniques.

Structural and dynamic determinants of ligand binding in the ternary complex of chicken liver bile acid binding protein with two bile salts revealed by NMR.

Bile acids are physiological detergents facilitating absorption, transport, and distribution of lipid-soluble vitamins and dietary fats;they also play a role as signaling molecules that activate nuclear receptors and regulate cholesterol metabolism. Bile acid circulation is mediated by bile acid binding proteins (BABPs), and a detailed structural study of the complex of BABPs with bile salts has become a key issue for the complete understanding of the role of these proteins and their involvement in cholesterol homeostasis. The solution structure here reported describes, at variance with previously determined singly ligated structures, a BABP in a ternary complex with two bile acid molecules, obtained by employing a variety of NMR experiments. Exchange processes between the two bound chenodeoxycholate molecules as well as the more superficial ligand and the free pool have been detected through ROESY and diffusion experiments. Significant backbone flexibility has been observed in regions located at the protein open end, facilitating bile salts exchange. A detailed description of the protonation states and tautomeric forms of histidines strongly supports the view that histidine protonation modulates backbone flexibility and regulates ligand binding. This structure opens the way to targeted site-directed mutagenesis and interaction studies to investigate both binding and nuclear localization mechanisms.

NMR dynamic studies suggest that allosteric activation regulates ligand binding in chicken liver bile acid-binding protein.

Apo chicken liver bile acid-binding protein has been structurally characterized by NMR. The dynamic behavior of the protein in its apo- and holo-forms, complexed with chenodeoxycholate, has been determined via (15)N relaxation and steady state heteronuclear (15)N((1)H) nuclear Overhauser effect measurements. The dynamic parameters were obtained at two pH values (5.6 and 7.0) for the apoprotein and at pH 7.0 for the holoprotein, using the model free approach. Relaxation studies, performed at three different magnetic fields, revealed a substantial conformational flexibility on the microsecond to millisecond time scales, mainly localized in the C-terminal face of the beta-barrel. The observed dynamics are primarily caused by the protonation/deprotonation of a buried histidine residue, His(98), located on this flexible face. A network of polar buried side chains, defining a spine going from the E to J strand, is likely to provide the long range connectivity needed to communicate motion from His(98) to the EF loop region. NMR data are accompanied by molecular dynamics simulations, suggesting that His(98) protonation equilibrium is the triggering event for the modulation of a functionally important motion, i.e. the opening/closing at the protein open end, whereas ligand binding stabilizes one of the preexisting conformations (the open form). The results presented here, complemented with an analysis of proteins belonging to the intracellular lipid-binding protein family, are consistent with a model of allosteric activation governing the binding mechanism. The functional role of this mechanism is thoroughly discussed within the framework of the mechanism for the enterohepatic circulation of bile acids.

Determinants of protein stability and folding: comparative analysis of beta-lactoglobulins and liver basic fatty acid binding protein.

A new energy decomposition approach, aimed at identifying residues playing a folding key role, has been applied here to three homologous proteins, belonging to the calycin superfamily, namely bovine and porcine beta-lactoglobulins and Liver basic fatty acid binding protein, sharing the same beta-barrel fold and different degree of sequence identities. All-atom, explicit solvent molecular dynamics simulations around the native conformation were used to generate, for each of the three proteins, energy maps which were further simplified through eigenvalue decomposition. Analysis of the components of the eigenvector associated with the lowest eigenvalue singled out those residues (hot sites) behaving as strongly interacting and possible nucleation centers. The results fit well with experimental folding data and, especially, with the analysis of side chain-side chain interaction conservation.

Heterologous expression of bovine and porcine beta-lactoglobulins in Pichia pastoris: towards a comparative functional characterisation.

Bovine and porcine beta-lactoglobulins were cloned and expressed in host cells with the aim of developing the tools necessary for their structural, functional and conformational characterisation by NMR techniques. Both lipocalins were expressed in Pichia pastoris, where the use of a constitutive promoter turned out to allow the highest productivity. The yield of recombinant proteins was further improved through multiple integration of the encoding genes and by increasing aeration of the transformed cultures. Both proteins were obtained in the culture medium at the concentration of 200 microg/ml. Recombinant lipocalins were purified by ion-exchange chromatography from the culture medium. A preliminary NMR characterisation showed that both proteins were correctly folded.

Effects of the Medicago scutellata trypsin inhibitor (MsTI) on cisplatin-induced cytotoxicity in human breast and cervical cancer cells.

BACKGROUND: Snail medic (Medicago scutellata L.) seeds exhibit a significantly higher content of a trypsin inhibitor than other Medicago species. This inhibitor belongs to the Bowman-Birk family of serine protease inhibitors (BBI) and exhibits a good sequence homology with the BBI from soybean, while presenting some differences. It has been suggested that BBIs have antitumoral and radio-protective activity. MATERIALS AND METHODS: In order to assess whether the inhibitor from Medicago scutellata (MsTI) seeds show similar properties to those of BBI from soybean with respect to potentiation of cisplatin-induced cytotoxicity, we evaluated the effects of MsTI on cisplatin-induced cell killing in MCF7 human breast carcinoma cells and HeLa human cervical carcinoma cells. RESULTS: The 24-hour treatment of MsTI in the cell culture medium decreased the clonogenic survival of MCF7 and HeLa cells in a dose-dependent manner and enhanced cisplatin-induced cytotoxicity. The presence of MsTI during the entire incubation period reduced the D37 of cisplatin by 40% in both the cell lines. CONCLUSION: MsTI could be an useful agent for the potentiation of cisplatin-mediated cancer treatment.

NMR solution structure of viscotoxin C1 from Viscum album species Coloratum ohwi: toward a structure-function analysis of viscotoxins.

The high resolution three-dimensional structure of the newly discovered plant viscotoxin C1, from the Asiatic Viscum album ssp. Coloratum ohwi, has been determined in solution by (1)H NMR spectroscopy at pH 3.6 and 285 K. The viscotoxin C1-fold, consisting of a helix-turn-helix motif and a short stretch of an antiparralel beta-sheet is very similar to that found for the highly similar viscotoxins A2 and A3 and for other related thionins. Different functional properties of members of the thionin family are discussed here in light of the structural and electrostatic properties. Among the very homologous family of alpha- and beta-thionins, known for their antimicrobial activity, the viscotoxin subfamily differs from the other members because of its high toxicity against tumoral cells. Key residues for the modulation of viscotoxin cytotoxicity have been identified on the basis of sequence and structural alignment.

[Self-esteem in adolescents. A study]. / L'autostima negli adolescenti. Uno studio.

In the present article the adolescents, self esteem is considered. An investigation has been effected in two schools of the Capital. Results show a strong relationship between Nursing and the problems related to the adolescence. Nursing answer and activities have to be expounded in the considered reality in order to have a nurses taking care of adolescents not only inside the hospital but also in the community. It is the case to develop a new model of nursing, an educational model related to health and wellness.

EF loop conformational change triggers ligand binding in beta-lactoglobulins.

Beta-lactoglobulins, belonging to the lipocalin family, are a widely studied group of proteins, characterized by the ability to solubilize and transport hydrophobic ligands, especially fatty acids. Despite many reports, the mechanism of ligand binding and the functional role of these proteins is still unclear, and many contradicting concepts are often encountered in the literature. In the present paper the comparative analysis of the binding properties of beta-lactoglobulins has been performed using sequence-derived information, structure-based electrostatic calculations, docking simulations, and NMR experiments. Our results reveal for the first time the mechanism of beta-lactoglobulin ligand binding, which is completely determined by the opening-closing of EF loop, triggered by Glu89 protonation. The alkaline shift observed for Glu89 pKa in porcine beta-lactoglobulin (pKa 9.7) with respect to the bovine species (pKa 5.5) depends upon the interplay of electrostatic effects of few nearby key residues. Porcine protein is therefore able to bind fatty acids provided that the appropriate pH solution conditions are met (pH > 8.6), where the EF loop conformational change can take place. The unusually high pH of binding detected for porcine beta-lactoglobulin seems to be functional to lipases activity. Theoretical pKa calculations extended to representative beta-lactoglobulins allowed the identification of key residues involved in structurally and functionally important electrostatic interactions. The results presented here provide a strong indication that the described conformational change is a common feature of all beta-lactoglobulins.

Competitive binding of fatty acids and the fluorescent probe 1-8-anilinonaphthalene sulfonate to bovine beta-lactoglobulin.

The use of spectroscopy in the study of fatty acids binding to bovine beta-lactoglobulin (BLG) appears to be a difficult task, as these acid compounds, assumed as the protein natural ligands, do not exhibit favorable optical response such as, for example, absorption or fluorescence. Therefore, the BLG fatty-acid equilibrium has been tackled by exploiting the competition between fatty acids and ANS, a widely used fluorescent hydrophobic probe, whose binding sites on the protein have been characterized recently. Two lifetime decays of the ANS-BLG complex have been found; the longer one has been attributed to the internal binding site and the shorter one to the external site. At increasing fatty acids concentration, the fractional weight associated with ANS bound to the internal site drops, in agreement with a model describing the competition of the dye with fatty acids, whereas the external site occupancy appears to be unaffected by the fatty acids binding to BLG. This model is supported by docking studies. An estimate of the acid-binding affinities for BLG has been obtained by implementing the fitting of the bound ANS intensities with a competitive binding model. A relevant dependence has been found upon the solution pH, in the range from 6 to 8, which correlates with the calyx accessibility modulated by the conformation of the EF loop. Fatty acids with longer aliphatic chains (palmitate and laurate) are found to display larger affinities for the protein and the interaction free energy nicely correlates with the number of contacts inside the protein calyx, in agreement with docking simulations.

Solution structure of chicken liver basic fatty acid binding protein.

Chicken liver basic fatty acid binding protein (Lb-FABP) belongs to the basic-type fatty acid binding proteins, a novel group of proteins isolated from liver of different non mammalian species whose structure is not known. The structure of Lb-FABP has been solved by (1)H NMR. The overall fold of Lb-FABP, common to the other proteins of the family, consists of ten antiparallel beta-strands organised in two nearly ortogonal beta-sheets with two alpha helices closing the protein cavity where small hydrophobic ligands can be bound. The binding specificity of the protein is not known, however, based on the high sequence and structural similarity with an orthologous protein, ileal lipid binding protein, it is suggested that bile acids may be the putative ligands.

Peptide models of folding initiation sites of bovine beta-lactoglobulin: identification of nativelike hydrophobic interactions involving G and H strands.

In an attempt to characterize the early folding events in bovine beta-lactoglobulin (BLG), a set of peptides, covering the flexible N-terminal region and the stable C-terminus beta-core, was synthesized and analyzed by circular dichroism and by nuclear magnetic resonance in water, trifluoroethanol (TFE), and sodium dodecyl sulfate (SDS) below and above the critical micellar concentration. The role of local and long-range hydrophobic interactions in guiding the folding has been investigated. For the peptide fragment covering the more flexible N-terminal region of BLG (beta-strands A, B), where both theoretical predictions and kinetic refolding experiments suggested the formation of non-native alpha-helix, no native long-range contacts were identified, and a helical secondary structure was stabilized only in the presence of 25 mM SDS. At variance, in 50% (v/v) TFE, native, long-range hydrophobic interactions were observed in the peptide covering the core region comprising G and H beta-strands. The side chains involved in these interactions form a nativelike hydrophobic cluster, thus suggesting that the GH region may act as the folding initiation site for BLG. This result is reinforced by the identification, in the urea denaturated BLG, of residual structure located at the level of the GH interface, as evidenced by NMR analysis. These results, in excellent agreement with kinetic, thermodynamic, and cold denaturation folding data, once more underline the utmost importance of the GH region for the stability and folding of BLG. Severe aggregation effects prevented the structural analysis of the peptide covering the EFGH region, indicating that this larger segment does not represent an independent folding domain and that the terminal alpha-helix is necessary for stabilizing the BLG folding core.