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.

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.

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.

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.

Conjugation of gold nanoparticles with multidentate surfactants for enhanced stability and biological properties.

This work originated from the need to functionalize surfactant-coated inorganic nanoparticles for biomedical applications, a process that is limited by excess unbound surfactant. These limitations are connected to the bioconjugation of targeting molecules that are often in equilibrium between the free aliquot in solution and that which binds the surface of the nanoparticles. The excess in solution can play a role in the biocompatability in vitro and in vivo of the final nanoparticles stock. For this purpose, we tested the ability of common surfactants - monothiolated polyethylene glycol and amphiphilic polymers - to colloidally stabilize nanoparticles as excess surfactant is removed and compared them to newly appearing multidentate surfactants endowed with high avidity for inorganic nanoparticles. Our results showed that monothiolated polyethylene glycol or amphiphilic polymers have an insufficient affinity to the nanoparticles and as the excess surfactant is removed the colloidal stability is lost, while multidentate high-avidity surfactants excel in the same regard, possibly allowing improvement in an array of nanoparticle applications, especially in those stated.

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.

A prospective study assessing the pattern of response of local disease at DCE-MRI after salvage radiotherapy for prostate cancer.

Background: To assess the pattern of response of presumed local lesions at dynamic contrast enhancement magnetic resonance imaging (DCE-MRI) after salvage radiotherapy (sRT). Methods: This is a prospective study conducted at a single Institution accruing patients with one or more local failures at DCE-MRI after radical prostatectomy between August 2017 and June 2020. Patients underwent exclusive sRT delivering 66-69 Gy and 73.5 Gy in 30 fractions to the whole prostatic fossa and to the local failure(s) seen at DCE-MRI, respectively.Patients were offered DCE-MRI at 3 months intervals after sRT until complete disappearance (CR) of the lesion(s) or up to a maximum of 4 revaluations. Results: 62 patients with 72 nodules were enrolled. All patients underwent the 1st revaluation, and 33 patients (53.2%) showed a CR. The median time to CR was 4.7 months. Four patients did not undergo further testing before achieving a CR and even considering these patients as no responses, the vast majority (87.1%, 95%CI: 78.5-94.4%) of lesions would have completely disappeared by 12 months from the end of sRT.The volume of the lesion at pre-sRT DCE-MRI was an independent predictor of CR at the 1st revaluation (OR: 0.076, 95%CI: 0.009-0.667; p = 0.020) along with time elapsed from sRT (OR: 3.399, 95% CI: 1.156-9.993, p = 0.026). Conclusions: The present study documents the complete disappearance of the vast majority of local lesions after dose-escalated sRT though this requires several months after sRT; timing of CR is at least in part predictable based on the volume of the lesion.Trial registration: Clinicaltrials.gov NCT04703543, registered July 15 2020, retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT04703543.

Fluorescence of KCl Aqueous Solution: A Possible Spectroscopic Signature of Nucleation.

Ion pairing in water solutions alters both the water hydrogen-bond network and ion solvation, modifying the dynamics and properties of electrolyte water solutions. Here, we report an anomalous intrinsic fluorescence of KCl aqueous solution at room temperature and show that its intensity increases with the salt concentration. From the ab initio density functional theory (DFT) and time-dependent DFT modeling, we propose that the fluorescence emission could originate from the stiffening of the hydrogen bond network in the hydration shell of solvated ion-pairs that suppresses the fast nonradiative decay and allows the slower radiative channel to become a possible decay pathway. Because computations suggest that the fluorophores are the local ion-water structures present in the prenucleation phase, this band could be the signature of the incoming salt precipitation.

A Prospective Study Assessing the Post-Prostatectomy Detection Rate of a Presumed Local Failure at mpMR with Either 64CuCl2 or 64CuPSMA PET/CT.

BACKGROUND: We aimed assess the detection rate (DR) of positron emission tomography/computed tomography with two novel tracers in patients referred for salvage radiotherapy (sRT) with a presumed local recurrence at multiparametric magnetic resonance (mpMR) after radical prostatectomy (RP). METHODS: The present prospective study was conducted at a single institution between August 2017 and June 2020. Eligibility criteria were undetectable PSA after RP; subsequent biochemical recurrence (two consecutive PSA rises to 0.2 ng/mL or greater); a presumed local failure at mpMR; no distant metastases at 18F-fluorocholine PET/CT (CH/PET); no previous history of androgen deprivation therapy. Patients were offered both 64CuCl2 PET/CT (CU/PET) and 64Cu-PSMA PET/CT (PSMA/PET) before sRT. After image co-registration, PET findings were compared to mpMR ones in terms of DR and independent predictors of DR investigated at logistic regression. RESULTS: A total of 62 patients with 72 nodules at mpMR were accrued. Compared to mpMR (DR = 100%, 95%CI: 94.9-100%), DRs were 47.2% (95%CI: 36.1-58.6%) and 54.4% (95%CI: 42.7-65.7%) for CU/PET and PSMA/PET, respectively (p < 0.001 for both). Both experimental PET/CT performed particularly poorly at PSA levels consistent with early sRT. CONCLUSIONS: The two novel radiotracers are inferior to mpMR in restaging the prostatic fossa for sRT planning purposes, particularly in the context of early salvage radiotherapy.

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.

Response on DCE-MRI predicts outcome of salvage radiotherapy for local recurrence after radical prostatectomy.

OBJECTIVE: To assess the predictive role of response on dynamic contrast enhancement on magnetic resonance imaging (DCE-MRI) of visible local lesions in the setting of salvage radiotherapy (sRT) after radical prostatectomy. METHODS: All patients referred for sRT for biochemical failure after radical prostatectomy from February 2014 to September 2016 were considered eligible if they had been restaged with DCE-MRI and had been found to have a visible lesion in the prostatic bed, but no distant/nodal disease on choline positron emission tomography (PET)-computed tomography (CT). Eligible patients were contacted during follow-up and offered reimaging with serial DCE-MRI until lesion resolution. Complete response (CR) was defined as the disappearance of the target lesion on DCE-MRI; prostate-specific antigen (PSA) recurrence was defined as a 0.2 ng/mL PSA rise above the nadir. Median follow-up after sRT was 41.5 months (range, 12.1-61.2 months). RESULTS: Fifty-nine patients agreed to undergo repeated DCE-MRI for a total of 64 studied lesions. Overall, 57 lesions (89.1%) showed a CR after 1 (51 patients) or 2 (6 patients) scans, while 7 lesions did not show any change (no response [NR]). At 42 months, no evidence of biochemical disease (bNED) survival was 74.7±6.4% and 64.3±21.0% for patients with CR and NR lesions, respectively (hazard ratio [HR], 3.181; 95% confidence interval [CI], 0.157-64.364; p = 0.451). When only patients treated with sRT without androgen deprivation were selected (n = 41), bNED survival rates at 42 months were 72.1±8.0% and 0, respectively (HR, 52.830; 95% CI, 1.893-1474.110; p = 0.020). CONCLUSIONS: Patients whose lesions disappear during follow-up have a better outcome than those with unchanged lesions after sRT alone.

Fusion US/MRI prostate biopsy using a computer aided diagnostic (CAD) system.

BACKGROUND: The aim of this study was to investigate the impact of computer aided diagnostic (CAD) system on the detection rate of prostate cancer (PCa) in a series of fusion prostate biopsy (FPB). METHODS: Two prospective transperineal FPB series (with or without CAD assistance) were analyzed and PCa detection rates compared with per-patient and per-target analyses. The χ2 and Mann-Whitney test were used to compare categorical and continuous variables, respectively. Univariable and multivariable regression analyses were applied to identify predictors of any and clinically significant (cs) PCa detection. Subgroup analyses were performed after stratifying for PI-RADS Score and lesion location. RESULTS: Out of 183 FPB, 89 were performed with CAD assistance. At per-patient analysis the detection rate of any PCa and of cs PCa were 56.3% and 30.6%, respectively; the aid of CAD was negligible for either any PCa or csPCa detection rates (P=0.45 and P=0.99, respectively). Conversely in a per-target analysis, CAD-assisted biopsy had significantly higher positive predictive value (PPV) for any PCa versus MRI-only group (58% vs. 37.8%, P=0.001). PI-RADS Score was the only independent predictor of any and csPCa, either in per-patient or per-target multivariable regression analysis (all P<0.029). In a subgroup per-patient analysis of anterior/transitional zone lesions, csPCa detection rate was significantly higher in the CAD cohort (54.5%vs.11.1%, respectively; P=0.028), and CAD assistance was the only predictor of csPCa detection (P=0.013). CONCLUSIONS: CAD assistance for FPB seems to improve detection of csPCa located in anterior/transitional zone. Enhanced identification and improved contouring of lesions may justify higher diagnostic performance.

On the propagation of the OH radical produced by Cu-amyloid beta peptide model complexes. Insight from molecular modelling.

Oxidative stress and metal dyshomeostasis are considered as crucial factors in the pathogenesis of Alzheimer's disease (AD). Indeed, transition metal ions such as Cu(ii) can generate Reactive Oxygen Species (ROS) via O2 Fenton-like reduction, catalyzed by Cu(ii) coordinated to the Amyloid beta (Aß) peptide. Despite intensive effort, the mechanisms of ROS-induced molecular damage remain poorly understood. In the present paper, we investigate on the basis of molecular modelling computations the mechanism of OH radical propagation toward the Aß peptide, starting from the end-product of OH radical generation by Cu(ii)·Aß. We evaluate (i) the OH oxidative capacity, as well as the energetics of the possible Aß oxidation target residues, by quantum chemistry Density Functional Theory (DFT) on coordination models of Cu(ii)/OH/Aß and (ii) the motion of the OH˙ approaching the Aß target residues by classical Molecular Dynamics (MD) on the full peptide Cu(ii)/OH/Aß(1-16). The results show that the oxidative capacity of OH coordinated Cu(ii)Aß is significantly lower than that of the free OH radical and that propagation toward Aß Asp and His residues is favoured over Tyr residues. These results are discussed on the basis of the recent literature on in vitro Aß metal-catalyzed oxidation and on the possible implications for the AD oxidative stress mechanism.

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.

Anomalous Intrinsic Fluorescence of HCl and NaOH Aqueous Solutions.

The unique properties of liquid water mainly arise from its hydrogen bond network. The geometry and dynamics of this network play a key role in shaping the characteristics of soft matter, from simple solutions to biosystems. Here we report an anomalous intrinsic fluorescence of HCl and NaOH aqueous solutions at room temperature that shows important differences in the excitation and emission bands between the two solutes. From ab initio time-dependent density functional theory modeling we propose that fluorescence emission could originate from hydrated ion species contained in transient cavities of the bulk solvent. These cavities, which are characterized by a stiff surface, could provide an environment that, upon trapping the excited state, suppresses the fast nonradiative decay and allows the slower radiative channel to become a possible decay pathway.

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.

Copper reduction and dioxygen activation in Cu-amyloid beta peptide complexes: insight from molecular modelling.

Alzheimer's disease (AD) involves a number of factors including an anomalous interaction of copper with the amyloid peptide (Aß), inducing oxidative stress with radical oxygen species (ROS) production through a three-step cycle in which O2 is gradually reduced to superoxide, oxygen peroxide and finally OH radicals. The purpose of this work has been to investigate the reactivity of 14 different Cu(ii)-Aß coordination models with the aim of identifying on an energy basis (Density Functional Theory (DFT) and classical Molecular Dynamics (MD)) the redox competent form(s). Accordingly, we have specifically focused on the first three steps of the cycle, i.e. ascorbate binding to Cu(ii), Cu(ii) → Cu(i) reduction and O2 reduction to O2-. Compared to the recent literature, our results broaden the set of possible redox competent metallopeptide forms responsible for ROS production. Indeed, in addition to the three-coordinated species containing one His ligand, a N-terminal amine group and the carboxylate side chain of the Asp1 residue of Aß already proposed, we found two other Cu-Aß coordination modes involving two histidines.