Stacking Interactions and Flexibility of Human Telomeric Multimers.

G-quadruplexes (G4s) are helical four-stranded structures forming from guanine-rich nucleic acid sequences, which are thought to play a role in cancer development and malignant transformation. Most current studies focus on G4 monomers, yet under suitable and biologically relevant conditions, G4s undergo multimerization. Here, we investigate the stacking interactions and structural features of telomeric G4 multimers by means of a novel low-resolution structural approach that combines small-angle X-ray scattering (SAXS) with extremely coarse-grained (ECG) simulations. The degree of multimerization and the strength of the stacking interaction are quantitatively determined in G4 self-assembled multimers. We show that self-assembly induces a significant polydispersity of the G4 multimers with an exponential distribution of contour lengths, consistent with a step-growth polymerization. On increasing DNA concentration, the strength of the stacking interaction between G4 monomers increases, as well as the average number of units in the aggregates. We utilized the same approach to explore the conformational flexibility of a model single-stranded long telomeric sequence. Our findings indicate that its G4 units frequently adopt a beads-on-a-string configuration. We also observe that the interaction between G4 units can be significantly affected by complexation with benchmark ligands. The proposed methodology, which identifies the determinants that govern the formation and structural flexibility of G4 multimers, may be an affordable tool aiding in the selection and design of drugs that target G4s under physiological conditions.

Recent Theoretical Insights into the Oxidative Degradation of Biopolymers and Plastics by Metalloenzymes.

Molecular modeling techniques have become indispensable in many fields of molecular sciences in which the details related to mechanisms and reactivity need to be studied at an atomistic level. This review article provides a collection of computational modeling works on a topic of enormous interest and urgent relevance: the properties of metalloenzymes involved in the degradation and valorization of natural biopolymers and synthetic plastics on the basis of both circular biofuel production and bioremediation strategies. In particular, we will focus on lytic polysaccharide monooxygenase, laccases, and various heme peroxidases involved in the processing of polysaccharides, lignins, rubbers, and some synthetic polymers. Special attention will be dedicated to the interaction between these enzymes and their substrate studied at different levels of theory, starting from classical molecular docking and molecular dynamics techniques up to techniques based on quantum chemistry.

Polymorphism and Ligand Binding Modulate Fast Dynamics of Human Telomeric G-Quadruplexes.

Telomeric G-quadruplexes (G4s) are promising targets in the design and development of anticancer drugs. Their actual topology depends on several factors, resulting in structural polymorphism. In this study, we investigate how the fast dynamics of the telomeric sequence AG3(TTAG3)3 (Tel22) depends on the conformation. By using Fourier transform Infrared spectroscopy, we show that, in the hydrated powder state, Tel22 adopts parallel and mixed antiparallel/parallel topologies in the presence of K+ and Na+ ions, respectively. These conformational differences are reflected in the reduced mobility of Tel22 in Na+ environment in the sub-nanosecond timescale, as probed by elastic incoherent neutron scattering. These findings are consistent with the G4 antiparallel conformation being more stable than the parallel one, possibly due to the presence of ordered hydration water networks. In addition, we study the effect of Tel22 complexation with BRACO19 ligand. Despite the quite similar conformation in the complexed and uncomplexed state, the fast dynamics of Tel22-BRACO19 is enhanced compared to that of Tel22 alone, independently of the ions. We ascribe this effect to the preferential binding of water molecules to Tel22 against the ligand. The present results suggest that the effect of polymorphism and complexation on the G4 fast dynamics is mediated by hydration water.

Assessing the Performance of Non-Equilibrium Thermodynamic Integration in Flavodoxin Redox Potential Estimation.

Flavodoxins are enzymes that contain the redox-active flavin mononucleotide (FMN) cofactor and play a crucial role in numerous biological processes, including energy conversion and electron transfer. Since the redox characteristics of flavodoxins are significantly impacted by the molecular environment of the FMN cofactor, the evaluation of the interplay between the redox properties of the flavin cofactor and its molecular surroundings in flavoproteins is a critical area of investigation for both fundamental research and technological advancements, as the electrochemical tuning of flavoproteins is necessary for optimal interaction with redox acceptor or donor molecules. In order to facilitate the rational design of biomolecular devices, it is imperative to have access to computational tools that can accurately predict the redox potential of both natural and artificial flavoproteins. In this study, we have investigated the feasibility of using non-equilibrium thermodynamic integration protocols to reliably predict the redox potential of flavodoxins. Using as a test set the wild-type flavodoxin from Clostridium Beijerinckii and eight experimentally characterized single-point mutants, we have computed their redox potential. Our results show that 75% (6 out of 8) of the calculated reaction free energies are within 1 kcal/mol of the experimental values, and none exceed an error of 2 kcal/mol, confirming that non-equilibrium thermodynamic integration is a trustworthy tool for the quantitative estimation of the redox potential of this biologically and technologically significant class of enzymes.

A De Novo-Designed Type 3 Copper Protein Tunes Catechol Substrate Recognition and Reactivity.

De novo metalloprotein design is a remarkable approach to shape protein scaffolds toward specific functions. Here, we report the design and characterization of Due Rame 1 (DR1), a de novo designed protein housing a di-copper site and mimicking the Type 3 (T3) copper-containing polyphenol oxidases (PPOs). To achieve this goal, we hierarchically designed the first and the second di-metal coordination spheres to engineer the di-copper site into a simple four-helix bundle scaffold. Spectroscopic, thermodynamic, and functional characterization revealed that DR1 recapitulates the T3 copper site, supporting different copper redox states, and being active in the O2 -dependent oxidation of catechols to o-quinones. Careful design of the residues lining the substrate access site endows DR1 with substrate recognition, as revealed by Hammet analysis and computational studies on substituted catechols. This study represents a premier example in the construction of a functional T3 copper site into a designed four-helix bundle protein.

Role of fast dynamics in the complexation of G-quadruplexes with small molecules.

G-quadruplexes (G4s) formed by the human telomeric sequence AG3 (TTAG3)3 (Tel22) play a key role in cancer and aging. We combined elastic incoherent neutron scattering (EINS) and quasielastic incoherent neutron scattering (QENS) to characterize the internal dynamics of Tel22 G4s and to assess how it is affected by complexation with two standard ligands, Berberine and BRACO19. We show that the interaction with the two ligands induces an increase of the overall mobility of Tel22 as quantified by the mean squared displacements (MSD) of hydrogen atoms. At the same time, the complexes display a lower stiffness than G4 alone. Two different types of motion characterize the G4 nanosecond timescale dynamics. Upon complexation, an increasing fraction of G4 atomic groups participate in this fast dynamics, along with an increase in the relevant characteristic length scales. We suggest that the entropic contribution to the conformational free energy of these motions might be crucial for the complexation mechanisms.

Uncommon retroperitoneal tumour: follicular dendritic cell sarcoma.

INTRODUCTION: Retroperitoneal Follicular Dendritic Cell Sarcomas represents rare tumours with aggressive biologic behaviour. Accurate diagnosis requires a combination of both morphological and immunohistochemical analyses. PATIENTS AND METHODS: A 61-year-old man was referred to our Department with a left perinephric mass. Computed tomography scan showed a 5.5 cm circumscribed mass in front of the left renal vein abutting the first jejunal loop, with moderate heterogeneous contrast enhancement. Positron emission/computed tomography showed increased focal uptake in the lesion. RESULTS: A retroperitoneal tumor located behind the first jejunal loop was found at laparotomy, encompassing the superior mesenteric vessels. Excision with en-bloc segmental small bowel resection was performed. Morphological and immunohistochemical analyses were consistent with Follicular Dendritic Cell Sarcoma. CONCLUSIONS: Complete surgical resection in specialized multidisciplinary centers represents the treatment of choice for both primary or recurrent lesions since there is still no consensus on the role of adjuvant radio-chemotherapy.

Rational Design of Fe2 (µ-PR2 )2 (L)6 Coordination Compounds Featuring Tailored Potential Inversion.

It was recently discovered that some redox proteins can thermodynamically and spatially split two incoming electrons towards different pathways, resulting in the one-electron reduction of two different substrates, featuring reduction potential respectively higher and lower than the parent reductant. This energy conversion process, referred to as electron bifurcation, is relevant not only from a biochemical perspective, but also for the ground-breaking applications that electron-bifurcating molecular devices could have in the field of energy conversion. Natural electron-bifurcating systems contain a two-electron redox centre featuring potential inversion (PI), i. e. with second reduction easier than the first. With the aim of revealing key factors to tailor the span between first and second redox potentials, we performed a systematic density functional study of a 26-molecule set of models with the general formula Fe2 (µ-PR2 )2 (L)6 . It turned out that specific features such as i) a Fe-Fe antibonding character of the LUMO, ii) presence of electron-donor groups and iii) low steric congestion in the Fe's coordination sphere, are key ingredients for PI. In particular, the synergic effects of i)-iii) can lead to a span between first and second redox potentials larger than 700 mV. More generally, the "molecular recipes" herein described are expected to inspire the synthesis of Fe2 P2 systems with tailored PI, of primary relevance to the design of electron-bifurcating molecular devices.

H2 Activation in [FeFe]-Hydrogenase Cofactor Versus Diiron Dithiolate Models: Factors Underlying the Catalytic Success of Nature and Implications for an Improved Biomimicry.

Catalytic H2 oxidation has been dissected by means of DFT into the key steps common to the Fe2 unit of both the [FeFe]-hydrogenase cofactor and selected biomimics. The aim was to elucidate the molecular details underlying the very different performances of the two systems. We found that the better enzyme performance is based on a single iron atom that is maintained electron-poor, favoring H2 binding, although embedded within a highly electron-rich cofactor, ensuring a facile oxidation of the Fe2 -H2 adduct. This is due to 1) CN- coordinating to both iron atoms, due to their amphipathic Lewis acid/base properties, and 2) the 4Fe4S subunit further withdrawing electrons from the Fe2 core. Preserving a moderate electron deficiency at a single iron also helps the cofactor preserve hydride affinity, which favors H2 cleavage. Such valuable characteristics allow the biocatalyst to turnover close to equilibrium conditions. All previous biomimicry has shown, in contrast, the impossibility to properly balance the two apparently contrasting aforementioned requisites, although evident progress has been made by the H2 -ase community. Disclosure of the differences identified could inspire the design of novel biomimics, for instance, reconsidering the use of CN- in the catalyst architecture. Indeed, in the presence of bases normally employed in oxidative catalysis, undesired stable protonation at coordinated CN- , which affects the opposite process (proton reduction), could be overcome.

Interaction of the H-Cluster of FeFe Hydrogenase with Halides.

FeFe hydrogenases catalyze H2 oxidation and production using an "H-cluster", where two Fe ions are bound by an aza-dithiolate (adt) ligand. Various hypotheses have been proposed (by us and others) to explain that the enzyme reversibly inactivates under oxidizing, anaerobic conditions: intramolecular binding of the N atom of adt, formation of the so-called "Hox/inact" state or nonproductive binding of H2 to isomers of the H-cluster. Here, we show that none of the above explains the new finding that the anaerobic, oxidative, H2-dependent reversible inactivation is strictly dependent on the presence of Cl- or Br-. We provide experimental evidence that chloride uncompetitively inhibits the enzyme: it reversibly binds to catalytic intermediates of H2 oxidation (but not to the resting "Hox" state), after which oxidation locks the active site into a stable, saturated, inactive form, the structure of which is proposed here based on DFT calculations. The halides interact with the amine group of the H-cluster but do not directly bind to iron. It should be possible to stabilize the inhibited state in amounts compatible with spectroscopic investigations to explore further this unexpected reactivity of the H-cluster of hydrogenase.

Catalytic Mechanism of Fungal Lytic Polysaccharide Monooxygenases Investigated by First-Principles Calculations.

Lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes that facilitate the degradation of recalcitrant polysaccharides by the oxidative cleavage of glycosidic bonds. They are gaining rapidly increasing attention as key players in biomass conversion, especially for the production of second-generation biofuels. Elucidation of the detailed mechanism of the LPMO reaction is a major step toward the assessment and optimization of LPMO efficacy in industrial biotechnology, paving the way to utilization of sustainable fuel sources. Here, we used density functional theory calculations to study the reaction pathways suggested to date, exploiting a very large active-site model for a fungal AA9 LPMO and using a celloheptaose unit as a substrate mimic. We identify a copper oxyl intermediate as being responsible for H-atom abstraction from the substrate, followed by a rapid, water-assisted hydroxyl rebound, leading to substrate hydroxylation.

Photocatalytic Hydrogen Evolution Driven by [FeFe] Hydrogenase Models Tethered to Fluorene and Silafluorene Sensitizers.

It is successfully shown that photocatalytic proton reduction to dihydrogen in the presence of a sacrificial electron donor, such as trimethylamine (TEA) and ascorbate, can be driven by compact sensitizer-catalyst dyads, that is, dithiolate-bridged [FeFe] hydrogenase models tethered to organic sensitizers, such as fluorenes and silafluorenes (1 a-4 a). The sensitizer-catalyst dyads 1 a-4 a show remarkable and promising catalytic activities as well as enhanced stabilities during photocatalysis performed under UV-light irradiation. The photocatalysis was carried out both in non-aqueous and aqueous media. The latter experiments were performed by solubilizing the photocatalysts within micelles formed by either sodium dodecyl sulfate (SDS) or cetyltrimethylammonium bromide (CTAB). In this study a turnover number of 539 (7 h) is achieved under optimized conditions, which corresponds to an exceptionally high turnover frequency of 77 h-1 . Theoretical investigations as well as emission decay experiments were performed to understand the observed phenomena together with the mechanisms of photocatalytic H2 generation.

Mechanistic Insight into Electrocatalytic H2 Production by [Fe2(CN){µ-CN(Me)2}(µ-CO)(CO)(Cp)2]: Effects of Dithiolate Replacement in [FeFe] Hydrogenase Models.

DFT has been used to investigate viable mechanisms of the hydrogen evolution reaction (HER) electrocatalyzed by [Fe2(CN){µ-CN(Me)2}(µ-CO)(CO)(Cp)2] (1) in AcOH. Molecular details underlying the proposed ECEC electrochemical sequence have been studied, and the key functionalities of CN- and amino-carbyne ligands have been elucidated. After the first reduction, CN- works as a relay for the first proton from AcOH to the carbyne, with this ligand serving as the main electron acceptor for both reduction steps. After the second reduction, a second protonation occurs at CN- that forms a Fe(CNH) moiety: i.e., the acidic source for the H2 generation. The hydride (formally 2e/H+), necessary to the heterocoupling with H+ is thus provided by the µ-CN(Me)2 ligand and not by Fe centers, as occurs in typical L6Fe2S2 derivatives modeling the hydrogenase active site. It is remarkable, in this regard, that CN- plays a role more subtle than that previously expected (increasing electron density at Fe atoms). In addition, the role of AcOH in shuttling protons from CN- to CN(Me)2 is highlighted. The incompetence for the HER of the related species [Fe2{µ-CN(Me)2}(µ-CO)(CO)2(Cp)2]+ (2+) has been investigated and attributed to the loss of proton responsiveness caused by CN- replacement with CO. In the context of hydrogenase mimicry, an implication of this study is that the dithiolate strap, normally present in all synthetic models, can be removed from the Fe2 core without loss of HER, but the redox and acid-base processes underlying turnover switch from a metal-based to a ligand-based chemistry. The versatile nature of the carbyne, once incorporated in the Fe2 scaffold, could be exploited to develop more active and robust catalysts for the HER.

Photophysical Properties of S, Se and Te-Substituted Deoxyguanosines: Insight into Their Ability To Act as Chemotherapeutic Agents.

Guanine and guanosine derivatives have long been in use as anticancer drugs and recently have been proposed also as photosensitizers in photodynamic therapy. By means of density functional theory and its time-dependent formulation, the potential power as UVA chemotherapeutic agents has been investigated computing the photophysical properties (absorption spectra, excitation energies, and spin-orbit matrix elements) of sulfur, selenium, and tellurium-substituted deoxyguanosines. Different pathways for the population of the lowest triplet state have been considered. Results show that all the examined systems have the lowest triplet state lying above the energy required for the production of the highly cytotoxic excited molecular oxygen 1Δg and that the heavy atom effect ensures an efficient intersystem spin crossing.

Reactivity of the Excited States of the H-Cluster of FeFe Hydrogenases.

FeFe hydrogenases catalyze H2 oxidation and formation at an inorganic active site (the "H-cluster"), which consists of a [Fe2(CO)3(CN)2(dithiomethylamine)] subcluster covalently attached to a Fe4S4 subcluster. This active site is photosensitive: visible light has been shown to induce the release of exogenous CO (a reversible inhibitor of the enzyme), shuffle the intrinsic CO ligands, and even destroy the H-cluster. These reactions must be understood because they may negatively impact the use of hydrogenase for the photoproduction of H2. Here, we explore in great detail the reactivity of the excited states of the H-cluster under catalytic conditions by examining, both experimentally and using TDDFT calculations, the simplest photochemical reaction: the binding and release of exogenous CO. A simple dyad model can be used to predict which excitations are active. This strategy could be used for probing other aspects of the photoreactivity of the H-cluster.

On the generation of OH(·) radical species from H2O2 by Cu(I) amyloid beta peptide model complexes: a DFT investigation.

According to different studies, the interaction between amyloid ß-peptide (Aß) and copper ions could yield radical oxygen species production, in particular the highly toxic hydroxyl radical OH(·) that is suspected to contribute to Alzheimer's disease pathogenesis. Despite intensive experimental and computational studies, the nature of the interaction between copper and Aß peptide, as well as the redox reactivity of the system, are still matter of debate. It was proposed that in Cu(II) → Cu(I) reduction the complex Cu(II)-Aß could follow a multi-step conformational change with redox active intermediates that may be responsible for OH(·) radical production from H2O2 through a Fenton-like process. The purpose of this work is to evaluate, using ab initio Density Functional Theory computations, the reactivity of different Cu(I)-Aß coordination modes proposed in the literature, in terms of OH(·) production. For each coordination model, we considered the corresponding H2O2 adduct and performed a potential energy surface scan along the reaction coordinate of O-O bond dissociation of the peroxide, resulting in the production of OH(·) radical, obtaining reaction profiles for the evaluation of the energetic of the process. This procedure allowed us to confirm the hypothesis according to which the most populated Cu(I)-Aß two-histidine coordination is not able to perform efficiently H2O2 reduction, while a less populated three-coordinated form would be responsible for the OH(·) production. We show that coordination modes featuring a third nitrogen containing electron-donor ligand (an imidazole ring of an histidine residue is slightly favored over the N-terminal amine group) are more active towards H2O2 reduction.

Silicon-Heteroaromatic [FeFe] hydrogenase model complexes: insight into protonation, electrochemical properties, and molecular structures.

To learn from Nature how to create an efficient hydrogen-producing catalyst, much attention has been paid to the investigation of structural and functional biomimics of the active site of [FeFe]-hydrogenase. To understand their catalytic activities, the µ-S atoms of the dithiolate bridge have been considered as possible basic sites during the catalytic processes. For this reason, a series of [FeFe]-H2 ase mimics have been synthesized and characterized. Different [FeFe]-hydrogenase model complexes containing bulky Si-heteroaromatic systems or fluorene directly attached to the dithiolate moiety as well as their mono-PPh3 -substituted derivatives have been prepared and investigated in detail by spectroscopic, electrochemical, X-ray diffraction, and computational methods. The assembly of the herein reported series of complexes shows that the µ-S atoms can be a favored basic site in the catalytic process. Small changes in the (hetero)-aromatic system of the dithiolate moiety are responsible for large differences in their structures. This was elucidated in detail by DFT calculations, which were consistent with the experimental results.

DFT dissection of the reduction step in H2 catalytic production by [FeFe]-hydrogenase-inspired models: can the bridging hydride become more reactive than the terminal isomer?

Density functional theory has been used to study diiron dithiolates [HFe2(xdt)(PR3)n(CO)5-nX] (n = 0, 2, 4; R = H, Me, Et; X = CH3S(-), PMe3, NHC = 1,3-dimethylimidazol-2-ylidene; xdt = adt, pdt; adt = azadithiolate; pdt = propanedithiolate). These species are related to the [FeFe]-hydrogenases catalyzing the 2H(+) + 2e(-) ↔ H2 reaction. Our study is focused on the reduction step following protonation of the Fe2(SR)2 core. Fe(H)s detected in solution are terminal (t-H) and bridging (µ-H) hydrides. Although unstable versus µ-Hs, synthetic t-Hs feature milder reduction potentials than µ-Hs. Accordingly, attempts were previously made to hinder the isomerization of t-H to µ-H. Herein, we present another strategy: in place of preventing isomerization, µ-H could be made a stronger oxidant than t-H (E°µ-H > E°t-H). The nature and number of PR3 unusually affect ΔE°t-H-µ-H: 4PEt3 models feature a µ-H with a milder E° than t-H, whereas the 4PMe3 analogues behave oppositely. The correlation ΔE°t-H-µ-H ↔ stereoelectronic features arises from the steric strain induced by bulky Et groups in 4PEt3 derivatives. One-electron reduction alleviates intramolecular repulsions only in µ-H species, which is reflected in the loss of bridging coordination. Conversely, in t-H, the strain is retained because a bridging CO holds together the Fe2 core. That implies that E°µ-H > E°t-H in 4-PEt3 species but not in 4PMe3 analogues. Also determinant to observe E°µ-H > E°t-H is the presence of a Fe apical σ-donor because its replacement with a CO yields E°µ-H < E°t-H even in 4PEt3 species. Variants with neutral NHC and PMe3 in place of CH3S(-) still feature E°µ-H > E°t-H. Replacing pdt with (Hadt)(+) lowers E° but yields E°µ-H < E°t-H, indicating that µ-H activation can occur to the detriment of the overpotential increase. In conclusion, our results indicate that the electron richness of the Fe2 core influences ΔE°t-H-µ-H, provided that (i) the R size of PR3 must be greater than that of Me and (ii) an electron donor must be bound to Fe apically.

Excited state properties of diiron dithiolate hydrides: implications in the unsensitized photocatalysis of H2 evolution.

Density functional theory (DFT) and time-dependent DFT (TDDFT) have been used to investigate how visible light photons can excite an asymmetrically substituted diiron hydride, [Fe2(pdt)(µ-H)(CO)4dppv](+) (1(+), dppv = cis-1,2-C2H2(PPh2)2; pdt = 1,3-propanedithiolate), as well as the symmetric species [Fe2(pdt)(µ-H)(CO)4(PMe3)2](+) (2(+)), which are the first photocatalysts of proton reduction operating without employing sensitizers (Wang, W.; Rauchfuss, T. B.; Bertini, L.; Zampella, G.; J. Am. Chem. Soc., 2012, 134, 4525). Theoretical results illustrate that the peculiar reactivity associated to the excited states of 1(+) and 2(+) is compatible with three different scenarios: (i) it can arise from the movement of the hydride ligand from fully bridging to semibridging/terminal coordination, which is expected to be more reactive toward protons; (ii) reactivity could be related to cleavage of a Fe-S bond, which implies formation of a transient Fe penta-coordinate species that would trigger a facile turnstile hydride isomerization, if lifetime excitation is long enough; (iii) also in line with a Fe-S bond cleavage is the possibility that after excited state decay, a highly basic S center is protonated so that a species simultaneously containing S-H(δ+) and Fe-H(δ-) moieties is formed and, once reduced by a suitable electron donor, it can readily afford H2 plus an unprotonated form of the FeFe complex. This last possibility is consistent with (31)P NMR and IR solution data. All the three possibilities are compatible with the capability of 1(+) and 2(+) to perform photocatalysis of hydrogen evolving reaction (HER) without sensitizer. Moreover, even though it turned out difficult to discriminate among the three scenarios, especially because of the lack of experimental excitation lifetimes, it is worth underscoring that all of the three pathways represent a novelty regarding diiron carbonyl photoreactivity, which is usually associated with CO loss. Results provide also a rationale to the experimental observations which showed that the simultaneous presence of donor ligands (dppv in the case of 1(+)) and a H ligand in the coordination environment of diiron complexes is a key factor to prevent CO photodissociation and catalyze HER. Finally, the comparison of photoexcitation behavior of 1(+) and 2(+) allows a sort of generalization about the functioning of such hydride species.

The Prognostic Value of DCE-MRI Findings before Salvage Radiotherapy after Radical Prostatectomy.

BACKGROUND: To investigate the predictive role of dynamic contrast-enhanced-magnetic resonance imaging (DCE-MRI) findings before salvage radiotherapy after radical prostatectomy (RP). METHODS: This retrospective study selected patients with biochemical failure (BF) after RP restaged with DCE-MRI. Patients underwent sRT in 30 fractions delivering 66-69 Gy and 73.5 Gy to the prostatic fossa and to the local failure as per DCE-MRI, respectively. Pelvic nodes were treated to 54 Gy in selected patients. The endpoint was BF after sRT. RESULTS: In total, 236 patients were analyzed and 146 (61.9%) had presumed local failure at DCE-MRI: 54.8%, 23.8% and 21.4% were found at the vesico-urethral anastomosis (VUA), the bladder neck and the retro-vesical space, respectively. The presence of a local failure at DCE-MRI halved the risk of BF; VUA-only location and lesion volume were independently correlated with survival without evidence of biochemical failure (bNED) at multivariable analysis. For patients with VUA-only disease up to 0.4 cc, the 4-year-bNED was 94.6% (95%CI: 80.2-98.6%) as opposed to 80.9% (95%CI: 71.6-87.4%) and 73.7% (95%CI: 63.1-81.8%) for other lesions and no macrodisease, respectively. CONCLUSIONS: DCE-MRI at restaging for BF after RP provides predictive and therapeutic information. Patients with small lesions at the VUA have an excellent prognosis after sRT.