Insight into the Mechanism of Action and Peptide-Membrane Interactions of Aib-Rich Peptides: Multitechnique Experimental and Theoretical Analysis.

The increase in resistant bacterial strains necessitates the identification of new antimicrobial molecules. Antimicrobial peptides (AMPs) are an attractive option because of evidence that bacteria cannot easily develop resistance to AMPs. The peptaibols, a class of naturally occurring AMPs, have shown particular promise as antimicrobial drugs, but their development has been hindered by their mechanism of action not being clearly understood. To explore how peptaibols might interact with membranes, circular dichroism, vibrational circular dichroism, linear dichroism, Raman spectroscopy, Raman optical activity, neutron reflectivity and molecular dynamics simulations have been used to study a small library of peptaibol mimics, the Aib-rich peptides. All the peptides studied quickly partitioned and oriented in membranes, and we found evidence of chiral interactions between the phospholipids and membrane-embedded peptides. The protocols presented in this paper open new ground by showing how chiro-optical spectroscopies can throw light on the mechanism of action of AMPs.

Helical Foldamers Incorporating Photoswitchable Residues for Light-Mediated Modulation of Conformational Preference.

An E unsaturated fumaramide linkage may be introduced into Aib peptide foldamer structures by standard coupling methods and photoisomerized to its Z (maleamide) isomer by irradiation with UV light. As a result of the photoisomerization, a new hydrogen-bonded contact becomes possible between the peptide domains located on either side of the unsaturated linkage. Using the fumaramide/maleamide linker to couple a chiral and an achiral fragment allows the change in hydrogen bond network to communicate a conformational preference, inducing a screw sense preference in the achiral domain of the maleamide-linked foldamers that is absent from the fumaramides. Evidence for the induced screw sense preference is provided by NMR and CD, and also by the turning on by light of the diastereoselectivity of a peptide chain extension reaction. The fumaramide/maleamide linker thus acts as a "conformational photodiode" that conducts stereochemical information as a result of irradiation by UV light.

Tomotherapy PET-guided dose escalation: A dosimetric feasibility study for patients with malignant pleural mesothelioma.

AIM: The aim of this study was to investigate whether a safe escalation of the dose to the pleural cavity and PET/CT-positive areas in patients with unresectable malignant pleural mesothelioma (MPM) is possible using helical tomotherapy (HT). MATERIAL AND METHODS: We selected 12 patients with MPM. Three planning strategies were investigated. In the first strategy (standard treatment), treated comprised a prescribed median dose to the planning target volume (PTV) boost (PTV1) of 64.5 Gy (range: 56 Gy/28 fractions to 66 Gy/30 fractions) and 51 Gy (range: 50.4 Gy/28 fractions to 54 Gy/30 fractions) to the pleura PTV (PTV2). Thereafter, for each patient, two dose escalation plans were generated prescribing 62.5 and 70 Gy (2.5 and 2.8 Gy/fraction, respectively) to the PTV1 and 56 Gy (2.24 Gy/fraction) to the PTV2, in 25 fractions. Dose-volume histogram (DVH) constraints and normal tissue complication probability (NTCP) calculations were used to evaluate the differences between the plans. RESULTS: For all plans, the 95 % PTVs received at least 95 % of the prescribed dose. For all patients, it was possible to perform the dose escalation in accordance with the Quantitative Analysis of Normal Tissue Effects in the Clinic (QUANTEC) constraints for organs at risk (OARs). The average contralateral lung dose was < 8 Gy. NTCP values for OARs did not increase significantly compared with the standard treatment (p > 0.05), except for the ipsilateral lung. For all plans, the lung volume ratio was strongly correlated with the V20, V30, and V40 DVHs of the lung (p < 0.0003) and with the lung mean dose (p < 0.0001). CONCLUSION: The results of this study suggest that by using HT it is possible to safely escalate the dose delivery to at least 62.5 Gy in PET-positive areas while treating the pleural cavity to 56 Gy in 25 fractions without significantly increasing the dose to the surrounding normal organs.

Erratum to: Tomotherapy PET-guided dose escalation--A dosimetric feasibility study for patients with malignant pleural mesothelioma.

Unfortunately, erroneous author affiliations were published in the article "Tomotherapy PET-guided dose escalation ­ A dosimetric feasibility study for patients with malignant pleural mesothelioma". The correct list of author affiliations reads as follows: Angelo Maggio 1, Claudia Cutaia 1, Amalia Di Dia 1, Sara Bresciani 1, Anna Miranti 1, Matteo Poli 1, Elena Delmastro 2, Elisabetta Garibaldi 2, Pietro Gabriele 2 and Michele Stasi 1. 1: Medical Physics Department, Candiolo Cancer Institute ­ FPO, IRCCS, Turin, Italy. 2: Radiotherapy Department, Candiolo Cancer Institute ­ FPO, IRCCS, Turin, Italy. We apologize for any inconveniences caused.

Engineering the structure of an N-terminal ß-turn to maximize screw-sense preference in achiral helical peptide chains.

Oligomers of α-aminoisobutyric acid (Aib) are achiral peptides that typically adopt 310 helical conformations in which enantiomeric left- and right-handed conformers are, necessarily, equally populated. Incorporating a single protected chiral residue at the N-terminus of the peptide leads to induction of a screw-sense preference in the helical chain, which may be quantified (in the form of "helical excess") by NMR spectroscopy. Variation of this residue and its N-terminal protecting group leads to the conclusion that maximal levels of screw-sense preference are induced by bulky chiral tertiary amino acids carrying amide protecting groups or by chiral quaternary amino acids carrying carbamate protecting groups. Tertiary L-amino acids at the N-terminus of the oligomer induce a left-handed screw sense, while quaternary L-amino acids induce a right-handed screw sense. A screw-sense preference may also be induced from the second position of the chain, weakly by tertiary amino acids, and much more powerfully by quaternary amino acids. In this position, the L enantiomers of both families induce a right-handed screw sense. Maximal, and essentially quantitative, control is induced by an L-α-methylvaline residue at both positions 1 and 2 of the chain, carrying an N-terminal carbamate protecting group.

Thionoglycine as a multifunctional spectroscopic reporter of screw-sense preference in helical foldamers.

A single thionoglycine (glycine thioamide, -HNCH2C(=S)-) residue inserted into a peptide foldamer provides both a pair of germinal protons for use as a (1)H NMR stereochemical probe and a chromophore giving rise to a well defined Cotton effect in CD. Comparison of the response of these two features to a local helically chiral environment validates them as independent methods for quantifying the conformational screw-sense preference of a helical oligomer, in this case a peptide made of repeated Aib units. The sign of the Cotton effect provides a measure of the sign of the screw-sense preference, while both the chemical shift separation of the anisochronous signals of the glycine CH2 group and the magnitude of the Cotton effect give an estimate of the helicity excess of the oligomer. The thionoglycine unit is readily introduced synthetically by a thionation of a BocGlyAibOMe dipeptide.

The N-terminal nonapeptide of cephaibols A and C: a naturally occurring example of mismatched helical screw-sense control.

The N-terminal nonapeptide domain of the fungal nonribosomal peptide antibiotics cephaibol A and cephaibol C (AcPheAib4LeuIvaGly- Aib) is reported to adopt a right-handed helical conformation in the crystalline state. However, this conformation is at odds with the left-handed helicity observed in solution in related synthetic oligomers capped with Ac-L-PheAib4 fragments. We report the synthesis of four diastereoisomers of the cephaibol N-terminal nonapeptide, and show by NMR and CD spectroscopy that the peptide containing the chiral amino acids Phe and Leu in the naturally occurring relative configuration exists in solution as an interconverting mixture of helical screw-sense conformers. In contrast, the nonapeptide containing the unnatural relative configuration at Phe and Leu adopts a single, stable helical screw-sense, which is left handed when the N-terminal Phe residue is L and right-handed when the N-terminal Phe residue is D.

Left-handed helical preference in an achiral peptide chain is induced by an L-amino acid in an N-terminal type II ß-turn.

Oligomers of the achiral amino acid Aib adopt helical conformations in which the screw-sense may be controlled by a single N-terminal residue. Using crystallographic and NMR techniques, we show that the left- or right-handed sense of helical induction arises from the nature of the ß-turn at the N terminus: the tertiary amino acid L-Val induces a left-handed type II ß-turn in both the solid state and in solution, while the corresponding quaternary amino acid L-α-methylvaline induces a right-handed type III ß-turn.

Diastereotopic fluorine substituents as 19F NMR probes of screw-sense preference in helical foldamers.

Ligating simple amino alcohol or amino ester monomers containing enantiotopic fluorine substituents to the C-terminus of a helical peptide places the fluorine atoms in diastereotopic environments, and gives two distinct and easily identifiable signals in the (19)F NMR spectrum. In the case of a dynamically inverting helix built from achiral monomers, the chemical shift separation between the (19)F signals provides a simple means of analysing the ratio of screw-sense conformers in the oligomer, in cases where an asymmetric bias leads to a screw-sense preference.

Partial thioamide scan on the lipopeptaibiotic trichogin GA IV. Effects on folding and bioactivity.

Backbone modification is a common chemical tool to control the conformation of linear peptides and to explore potentially useful effects on their biochemical and biophysical properties. The thioamide, ψ[CS-NH], group is a nearly isosteric structural mimic of the amide (peptide) functionality. In this paper, we describe the solution synthesis, chemical characterization, preferred conformation, and membrane and biological activities of three, carefully selected, peptide analogues of the lipopeptaibiotic [Leu(11)-OMe] trichogin GA IV. In each analogue, a single thioamide replacement was incorporated. Sequence positions near the N-terminus, at the center, and near the C-terminus were investigated. Our results indicate that (i) a thioamide linkage is well tolerated in the overall helical conformation of the [Leu(11)-OMe] lipopeptide analogue and (ii) this backbone modification is compatible with the preservation of its typical membrane leakage and antibiotic properties, although somewhat attenuated.

Couplings between peptide linkages across a 3(10)-helical hydrogen bond revealed by two-dimensional infrared spectroscopy.

Vibrational couplings between the amide modes are keenly dependent on peptide structure. Site-specific couplings can inform us of molecular conformation in detail. For example, when an amide-I mode couples to an amide-II mode that is three residues away because they are brought into proximity in the presence of an intramolecular C=O...H-N hydrogen bond, the coupling can provide direct evidence for single helical turn formation, a proposed key step in coil-helix transition. In this work, we measure 2D IR spectra of a 3(10)-helical hexapeptide, Z-Aib-l-Leu-(Aib)(2)-Gly-Aib-OtBu, and its (13)C=(18)O-Leu monolabeled and (13)C=(18)O-Leu/(15)N-Gly bis-labeled isotopomers in CDCl(3). The isotope-dependent amide-I/II cross-peaks clearly reveal the existence of vibrational coupling between the second and fourth peptide linkages that are connected through a 3(10)-helical hydrogen bond. Our results demonstrate that the combination of 2D IR and (13)C=(18)O/(15)N labeling is a useful structural method for probing local peptide conformation with residue-level specificity.

Is the backbone conformation of C(alpha)-methyl proline restricted to a single region?

C(alpha)-methyl-L-proline, or L-(alphaMe)Pro, is probably the most conformationally constrained alpha-amino acid. In particular, its omega and phi torsion angles are restricted to about 180 and -60 degrees, respectively, and only three ranges of values are theoretically available for psi in mono- or longer peptides, namely, about -30 degrees (cis', 3(10)/alpha-helical structure), 60 degrees (inverse gamma turn), or 140 degrees (trans', poly(L-Pro)(n) II structure). In this work, we examined the tendency of a number of N(alpha)-acyl dipeptide N'-alkylamides of the type RCO-(alphaMe)Pro-Xxx-NHR' or RCO-Xxx-(alphaMe)Pro-NHR', in which Xxx is L (or D)-Ala, Aib (alpha-aminoisoburyric acid), or L (or D)-(alphaMe)Pro, long enough to fold into intramolecularly hydrogen-bonded gamma or beta turns. The results are compared with those obtained for the corresponding dipeptides based on Pro, a well-known turn-forming residue. For the crystal-state 3D-structural analysis we used X-ray diffraction, whereas our solution conformational analysis was heavily based on the FTIR absorption and (1)H and (13)C NMR spectroscopy techniques. We conclude that (alphaMe)Pro is able to explore both trans' and cis' psi areas of the conformational space, but in (alphaMe)Pro the latter is overwhelmingly more populated, in marked contrast to the Pro preference. This finding is a clear indication that in (alphaMe)Pro the major 3D-structural determinant is the C(alpha)-methyl group. The circular dichroism (CD) signature of a peptide type III' beta-turn conformation is also proposed.

Toward detecting the formation of a single helical turn by 2D IR cross peaks between the amide-I and -II modes.

We have combined two-dimensional infrared (2D IR) spectroscopy and isotope substitutions to reveal the vibrational couplings between a pair of amide-I and -II modes that are several residues away but directly connected through a hydrogen bond in a helical peptide. This strategy is demonstrated on a 3(10)-helical hexapeptide, Z-Aib-L-Leu-(Aib)2-Gly-Aib-OtBu, and its 13C=18O-Leu monolabeled and 13C=18O-Leu/15N-Gly bis-labeled isotopomers in CDCl3. The isotope-dependent amide-I/II cross peaks clearly show that the second and fourth peptide linkages are vibrationally coupled as they are in proximity, forming a 3(10)-helical turn. The experimental spectra are compared to simulations based on a vibrational exciton Hamiltonian model that fully takes into account the amide-I and -II modes. The amide-II local mode frequency is evaluated by a new model based on the effects of hydrogen-bond geometry and sites. Ab initio nearest-neighbor coupling maps of the amide-I/I, -I/II, -II/I and -II/II modes are generated by isotopically isolating the local modes of N-acetyl-glycine N'-methylamide (AcGlyNHMe). Longer range couplings are modeled by transition charge interactions. The effects of the capping groups are incorporated and isotope effects are analyzed based on ab initio calculations of six model compounds. The main features of the 2D IR spectra are reproduced by this modeling. The conformational sensitivity of the isotope-dependent amide-I/II cross peaks is discussed in comparison with the calculated spectra for a semiextended structure. Our experimental and theoretical study demonstrates that the combination of 2D IR and 13C=18O/15N labeling is a useful structural method for detecting helical turn formation with residue-level specificity.

PET-CT post therapy dosimetry in radioembolization with resin 90Y microspheres: Comparison with pre-treatment SPECT-CT 99mTc-MAA results.

Resin microspheres radioembolization is an effective treatment for liver tumors when the surgical option is not feasible. Doses delivered to tumor and normal liver can be assess in the pre-therapy phase by means of a 99mTc-MAA SPECT-CT simulation and after the treatment with 90Y PET-CT acquisition. The optimal therapeutic 90Y activity is determined on 99mTc-MAA SPECT-CT dose results in order to avoid healthy parenchyma toxicity and to effectively irradiate the tumor. The assumption of identical radiopharmaceutical distribution between simulation and verification is still under debate and literature data showed controversial results. In this study 10 HCC patient's dosimetry performed on 99mTc SPECT-CT and 90Y PET-CT were compared. Patients were selected when a good agreement between the pre and post-therapy distribution was observed in order to investigate the intrinsic dosimetric variations between the two imaging modalities. Mean doses (MIRD and Voxel approaches) showed a good correlation (Pearson's coefficient r > 0.90) both for tumor and normal liver. Dose Volume Histogram curves were compared with a good agreement particularly for normal liver (D50). Goal doses were achieved for 90% of patients. Bland-Altman analysis indicates lower variations for healthy parenchyma than for tumor (1.96 SD equal to 9.1 Gy and 68 Gy respectively) confirming the robustness of the dose-toxicity approach. PET-CT dosimetry well correlates with SPECT-CT doses (under assumption of same catheter position and 90Y activity). Better agreement was showed for 7/10 and 8/10 patients for T and NL respectively, confirming dosimetry as effective tool to optimize and individualize the treatment.

Performance evaluation of a new time of flight PET/CT scanner: Results of a multicenter study.

PURPOSE: The aim of this multicenter study was to evaluate the performance of the upgraded version of the Ingenuity TF PET/CT scanner, according to the NEMA NU-2 2012 standards. METHODS: Spatial resolution, sensitivity, count rate response, scatter fraction, image quality and accuracy were evaluated on three Ingenuity TF scanners installed in Italian hospitals. Furthermore, energy and timing resolution were measured. A detailed image quality phantom analysis was performed to evaluate the effect of different clinical reconstruction parameters, including the application of PSF correction. RESULTS: Results show an average spatial resolution of 4.7 mm and an average absolute system sensitivity of 7.9 cps/kBq. The average maximum NECR was 119.83 kcps at 20.67 kBq/ml, while the maximum true event rate was 322.62 kcps at the concentration of 24.51 kBq/ml. The average maximum bias below NECR peak was 12.58%. All the results of NEMA tests were in agreement with the values declared by the manufacturer. The estimated average energy and timing resolution were 10.83% and 536.2 ps, respectively. Image quality phantom analysis obtained with different reconstruction settings showed that PSF correction was the parameter that affected mainly on contrast recovery coefficient, while the iteration number and amplitude of Gaussian filter had no significant effect. Of relevance, the application of PSF correction never led to recovery coefficient values higher than 100% and to Gibbs or edge artifacts. CONCLUSIONS: The new Ingenuity TF model shows physical performance similar to other scanners of the latest generation for all standard NEMA NU2-2012 measurements.

Comparison of two different EPID-based solutions performing pretreatment quality assurance: 2D portal dosimetry versus 3D forward projection method.

PURPOSE: The aim of this paper is to characterize two different EPID-based solutions for pre-treatment VMAT quality assurance, the 2D portal dosimetry and the 3D projection technique. Their ability to catch the main critical delivery errors was studied. METHODS: Measurements were performed with a linac accelerator equipped with EPID aSi1000, Portal Dose Image Prediction (PDIP), and PerFRACTION softwares. Their performances were studied simulating perturbations of a reference plan through systematic variations in dose values and micromultileaf collimator position. The performance of PDIP, based on 2D forward method, was evaluated calculating gamma passing rate (%GP) between no-error and error-simulated measurements. The impact of errors with PerFRACTION, based on 3D projection technique, was analyzed by calculating the difference between reference and perturbed DVH (%ΔD). Subsequently pre-treatment verification with PerFRACTION was done for 27 patients of different pathologies. RESULTS: The sensitivity of PerFRACTION was slightly higher than sensitivity of PDIP, reaching a maximum of 0.9. Specificity was 1 for PerFRACTION and 0.6 for PDIP. The analysis of patients' DVHs indicated that the mean %ΔD was (1.2 ±â€¯1.9)% for D2%, (0.6 ±â€¯1.7)% for D95% and (-0.0 ±â€¯1.2)% for Dmean of PTV. Regarding OARs, we observed important discrepancies on DVH but that the higher dose variations were in low dose area (<10 Gy). CONCLUSIONS: This study supports the introduction of the new 3D forward projection method for pretreatment QA raising the claim that the visualization of the delivered dose distribution on patient anatomy has major advantages over traditional portal dosimetry QA systems.

Conformational photoswitching of a synthetic peptide foldamer bound within a phospholipid bilayer.

The dynamic properties of foldamers, synthetic molecules that mimic folded biomolecules, have mainly been explored in free solution. We report on the design, synthesis, and conformational behavior of photoresponsive foldamers bound in a phospholipid bilayer akin to a biological membrane phase. These molecules contain a chromophore, which can be switched between two configurations by different wavelengths of light, attached to a helical synthetic peptide that both promotes membrane insertion and communicates conformational change along its length. Light-induced structural changes in the chromophore are translated into global conformational changes, which are detected by monitoring the solid-state (19)F nuclear magnetic resonance signals of a remote fluorine-containing residue located 1 to 2 nanometers away. The behavior of the foldamers in the membrane phase is similar to that of analogous compounds in organic solvents.

A pre-treatment quality assurance survey on 384 patients treated with helical intensity-modulated radiotherapy.

The gamma index pass rate (%GP) of 384 helical Tomotherapy pre-patient quality assurance, acquired with ArcCHECK, is presented, analyzed, and correlated to plan characteristics. Average %GP was higher than 90% and correlated strongly with gamma method, irradiated length, pitch, maximum dose to diodes, and dose per fraction.

Experimental and theoretical spectroscopic study of 3(10)-helical peptides using isotopic labeling to evaluate vibrational coupling.

Coupling between the amide linkages in a peptide or protein is the key physical property that gives vibrational spectra and circular dichroism sensitivity to secondary structures. By use of (13)C isotopic labeling on individual and pairs of amide C═O groups, the amide I band for selected residues was effectively isolated in designed hexa- and octapeptides having dominant 3(10)-helical conformations. The resultant frequency and intensity responses were measured with IR absorption, vibrational circular dichroism (VCD), and Raman spectroscopies and simulated with density functional theory (DFT) based computations. Band fitting the spectral components and correlating the results to the computed coupling between selected labeled positions were used to determine coupling constant signs and to estimate their magnitudes for specific sequences. The observed frequency and intensity patterns, and their variation between IR and VCD with label position in the sequence, follow the theoretical predictions to a large degree, but are complicated by end effects that alter the local force field (FF) for some residues in these short peptides. These FF variations were overestimated in the theoretical models which may be evidence of structural variations not included in the model. By analyzing the simulations with different coupling models, the coupling constants were determined to lie in a range (positive) +3-5 cm(-1) for sequential residues (i,i+1) and with (negative) -3 cm(-1) as an upper bound for alternate ones (i,i+2). The sequential amide coupling for 3(10)-helices is weaker than for α-helices but has the same sign and is larger than and oppositely signed as compared to 3(1)-, or poly-(Pro)(n) type-II, helices.