Financial Toxicity in Renal Patients (FINTORE) Study: A Cross-Sectional Italian Study on Financial Burden in Kidney Disease-A Project Protocol.

Financial toxicity (FT) refers to the negative impact of health-care costs on clinical conditions. In general, social determinants of health, especially poverty, socioenvironmental stressors, and psychological factors, are increasingly recognized as important determinants of non-communicable diseases, such as chronic kidney disease (CKD), and their consequences. We aim to investigate the prevalence of FT in patients at different stages of CKD treated in our universal health-care system and from pediatric nephrology, hemodialysis, peritoneal dialysis and renal transplantation clinics. FT will be assessed with the Patient-Reported Outcome for Fighting Financial Toxicity (PROFFIT) score, which was first developed by Italian oncologists. Our local ethics committee has approved the study. Our population sample will answer the sixteen questions of the PROFFIT questionnaire, seven of which are related to the outcome and nine the determinants of FT. Data will be analyzed in the pediatric and adult populations and by group stratification. We are confident that this study will raise awareness among health-care professionals of the high risk of adverse health outcomes in patients who have both kidney disease and high levels of FT. Strategies to reduce FT should be implemented to improve the standard of care for people with kidney disease and lead to truly patient-centered care.

Evidence of a Distinctive Enantioselective Binding Mode for the Photoinduced Radical Cyclization of α-Chloroamides in Ene-Reductases.

We demonstrate here through molecular simulations and mutational studies the origin of the enantioselectivity in the photoinduced radical cyclization of α-chloroacetamides catalyzed by ene-reductases, in particular the Gluconobacter oxidans ene-reductase and the Old Yellow Enzyme 1, which show opposite enantioselectivity. Our results reveal that neither the π-facial selectivity model nor a protein-induced selective stabilization of the transition states is able to explain the enantioselectivity of the radical cyclization in the studied flavoenzymes. We propose a new enantioinduction scenario according to which enantioselectivity is indeed controlled by transition-state stability; however, the relative stability of the prochiral transition states is not determined by direct interaction with the protein but is rather dependent on an inherent degree of freedom within the substrate itself. This intrinsic degree of freedom, distinct from the traditional π-facial exposure mode, can be controlled by the substrate conformational selection upon binding to the enzyme.

The electron-proton bottleneck of photosynthetic oxygen evolution.

Photosynthesis fuels life on Earth by storing solar energy in chemical form. Today's oxygen-rich atmosphere has resulted from the splitting of water at the protein-bound manganese cluster of photosystem II during photosynthesis. Formation of molecular oxygen starts from a state with four accumulated electron holes, the S4 state-which was postulated half a century ago1 and remains largely uncharacterized. Here we resolve this key stage of photosynthetic O2 formation and its crucial mechanistic role. We tracked 230,000 excitation cycles of dark-adapted photosystems with microsecond infrared spectroscopy. Combining these results with computational chemistry reveals that a crucial proton vacancy is initally created through gated sidechain deprotonation. Subsequently, a reactive oxygen radical is formed in a single-electron, multi-proton transfer event. This is the slowest step in photosynthetic O2 formation, with a moderate energetic barrier and marked entropic slowdown. We identify the S4 state as the oxygen-radical state; its formation is followed by fast O-O bonding and O2 release. In conjunction with previous breakthroughs in experimental and computational investigations, a compelling atomistic picture of photosynthetic O2 formation emerges. Our results provide insights into a biological process that is likely to have occurred unchanged for the past three billion years, which we expect to support the knowledge-based design of artificial water-splitting systems.

[Tic douloureux sustained by an eye tumor].

Cancer is a major cause of morbidity and mortality in solid organ transplantation. Nonmelanoma skin cancer (NMSC) such as basocellular (BCC) and spinocellular (SCC) carcinoma, are common in renal transplant recipients. We report a case of an SCC affecting a lacrimal gland in a subject with kidney transplantation. A man aged 75 years who had suffered from glomerulopathy since 1967 and subsequently started haemodialysis, in 1989 was transplanted from a living donor. In 2019, he suffered paresthesia and pain in his right eyebrow arch and he was diagnosed to have neuralgia of the fifth cranial nerve. The failure of medical treatment and the development of a mass in his eyelid plus exophthalmos induced healthcare professionals to perform a magnetic resonance. The latter showed a retrobulbar mass measuring 39×22×16 mm3. Biopsy revealed an SCC and the patient underwent eye exenteration. Although NMSC of the eye is an extremely rare condition, risk factors such as male sex, history of glomerulopathy, and duration of immunosuppression should be taken into consideration at the time of the onset of eye symptoms.

Alternative Fast and Slow Primary Charge-Separation Pathways in Photosystem II.

Photosystem-II (PSII) is a multi-subunit protein complex that harvests sunlight to perform oxygenic photosynthesis. Initial light-activated charge separation takes place at a reaction centre consisting of four chlorophylls and two pheophytins. Understanding the processes following light excitation remains elusive due to spectral congestion, the ultrafast nature, and multi-component behaviour of the charge-separation process. Here, using advanced computational multiscale approaches which take into account the large-scale configurational flexibility of the system, we identify two possible primary pathways to radical-pair formation that differ by three orders of magnitude in their kinetics. The fast (short-range) pathway is dominant, but the existence of an alternative slow (long-range) charge-separation pathway hints at the evolution of redundancy that may serve other purposes, adaptive or protective, related to formation of the unique oxidative species that drives water oxidation in PSII.

Molecular dynamics simulations and kinetic measurements provide insights into the structural requirements of substrate size-dependent specificity of oligogalacturonide oxidase 1 (OGOX1).

Oligogalacturonides (OGs) are pectin fragments released from the breakdown of the homogalacturonan during pathogenesis that act as Damage-Associated Molecular Patterns. OG-oxidase 1 (OGOX1) is an Arabidopsis berberine bridge enzyme-like (BBE-l) oligosaccharide oxidase that oxidizes OGs, impairing their elicitor activity and concomitantly releasing H2O2. The OG-oxidizing activity of OGOX1 is markedly pH-dependent, with optimum pH around 10, and is higher towards OGs with a degree of polymerization higher than two. Here, the molecular determinants of OGOX1 responsible for the binding of OGs with different lengths have been investigated through molecular dynamics simulations and enzyme kinetics studies. OGOX1 was simulated in complex with OGs with different degree of polymerization such as di-, tri-, tetra- and penta-galacturonide, in water solution at alkaline pH. Our simulations revealed that, among the four OGOX1/OG combinations, the penta-galacturonide (OG5) showed the best conformation in the active site to be efficiently oxidized by OGOX1. The optimal conformation can be stabilized by salt-bridges formed between the carboxyl groups of OG5 and five positively charged amino acids of OGOX1, highly conserved in all OGOX paralogs. Our results suggest that these interactions limit the mobility of OG5 as well as longer OGs, contributing to maintain the terminal monomer of OGs in the optimal orientation in order to be oxidized by the enzyme. In accordance with these results, the enzyme efficiency (Kcat/KM) of OGOX1 on OG5 (40.04) was found to be significantly higher than that on OG4 (13.05) and OG3 (0.6).

Evidence for a high pKa of an aspartic acid residue in the active site of CALB by a fully atomistic multiscale approach.

Candida antarctica Lipase B (CALB) is a paradigm for the family of lipases. At pH 7, the optimal pH for catalysis, the protonation state of an aspartic acid of the active site (Asp134) could not be conclusively assigned. In fact, the pKa estimate provided by a widely used computational tool, namely PropKa, that predicts pKa values of ionizable groups in proteins based on the crystallographic structure, is only slightly above 7 (pKa = 7.25). This, along with the lack of an experimental evaluation, makes the assignment of its protonation state at neutral pH challenging. Here, we calculate the pKa of Asp134 by means of a fully atomistic multiscale computational approach based on classical molecular dynamics (MD) simulation and the perturbed matrix method (PMM), namely the MD-PMM approach. MD-PMM is able to take into account the dynamics of the system and, at the same time, to treat the deprotonation step at the quantum level. The calculations provide a pKa value of 8.9 ± 1.1, hence suggesting that Asp134 in CALB should be protonated at neutral, and even at slightly basic, pH.Communicated by Ramaswamy H. Sarma.

A molecular dynamics-guided mutagenesis identifies two aspartic acid residues involved in the pH-dependent activity of OG-OXIDASE 1.

During the infection, plant cells secrete different OG-oxidase (OGOX) paralogs, defense flavoproteins that oxidize the oligogalacturonides (OGs), homogalacturonan fragments released from the plant cell wall that act as Damage Associated Molecular Patterns. OGOX-mediated oxidation inactivates their elicitor nature, but on the other hand makes OGs less hydrolysable by microbial endo-polygalacturonases (PGs). Among the different plant defense responses, apoplastic alkalinization can further reduce the degrading potential of PGs by boosting the oxidizing activity of OGOXs. Accordingly, the different OGOXs so far characterized showed an optimal activity at pH values greater than 8. Here, an approach of molecular dynamics (MD)-guided mutagenesis succeeded in identifying the amino acids responsible for the pH dependent activity of OGOX1 from Arabidopsis thaliana. MD simulations indicated that in alkaline conditions (pH 8.5), the residues Asp325 and Asp344 are engaged in the formation of two salt bridges with Arg327 and Lys415, respectively, at the rim of enzyme active site. According to MD analysis, the presence of such ionic bonds modulates the size and flexibility of the cavity used to accommodate the OGs, in turn affecting the activity of OGOX1. Based on functional properties of the site-directed mutants OGOX1.D325A and OGOX.D344A, we demonstrated that Asp325 and Asp344 are major determinants of the alkaline-dependent activity of OGOX1.

Mechanism of Oxygen Evolution and Mn4CaO5 Cluster Restoration in the Natural Water-Oxidizing Catalyst.

Water oxidation occurring in the first steps of natural oxygenic photosynthesis is catalyzed by the pigment/protein complex Photosystem II. This process takes place on the Mn4Ca cluster located in the core of Photosystem II and proceeds along the five steps (S0-S4) of the so-called Kok-Joliot cycle until the release of molecular oxygen. The catalytic cycle can therefore be started afresh through insertion of a new water molecule. Here, combining quantum mechanics/molecular mechanics simulations and minimum energy path calculations, we characterized on different spin surfaces the events occurring in the last sector of the catalytic cycle from structural, electronic, and thermodynamic points of view. We found that the process of oxygen evolution and water insertion can be described well by a two-step mechanism, with oxygen release being the rate-limiting step of the process. Moreover, our results allow us to identify the upcoming water molecule required to regenerate the initial structure of the Mn4Ca cluster in the S0 state. The insertion of the water molecule was found to be coupled with the transfer of a proton to a neighboring hydroxide ion, thus resulting in the reconstitution of the most widely accepted model of the S0 state.

Pivotal role of the redox-active tyrosine in driving the water splitting catalyzed by photosystem II.

Photosynthetic water oxidation is catalyzed by the Mn4Ca cluster in photosystem II (PSII). The nearby redox-active tyrosine (YZ) serves as a direct electron acceptor of the Mn4Ca cluster and it forms a low-barrier H-bond (LBHB) with a neighboring histidine residue (D1-His190). Experimental evidence indicates that YZ oxidation triggers changes in the hydrogen bonding network that precede proton abstraction from the Mn4Ca cluster. In order to characterize such changes, we compare ab initio molecular dynamics simulations of different states of the catalytic cycle of PSII with dynamics of isolated tyrosine models (namely, p-cresol) in different oxidation states. The systematic comparison of the H-bond networks in different simulated systems suggests that the YZ oxidation leads to a water hydration pattern which is more similar to that of the neutral p-cresol rather than that of the p-cresol anion. Our simulations also reveal the twofold nature of the interactions between YZ and the Mn4Ca cluster. Firstly, the YZ oxidation triggers rapid structural changes of the H-bond pattern in the proximity of the cluster which have been observed to propagate on the ps time scale on the Ca2+ hydration shell up to other water molecules in the proximity of the cluster. Secondly, it is clear that YZ interacts with the Mn4Ca cluster also through Coulombic interactions mediated by CP43-Arg357 through the remaining positive charge of the pair. Our results are able to identify, for the first time, the structural rearrangements guided by the oxidation of YZ necessary for the evolution of the water splitting reaction in PSII. Based on these findings, we propose a mechanism of structural changes which is functional towards the progression of the catalytic cycle in PSII.

On the comparison between differential vibrational spectroscopy spectra and theoretical data in the carboxyl region of photosystem II.

Understanding the structural modification experienced by the Mn4 CaO5 oxygen-evolving complex of photosystem II along the Kok-Joliot's cycle has been a challenge for both theory and experiments since many decades. In particular, differential infrared spectroscopy was extensively used to probe the surroundings of the reaction center, to catch spectral changes between different S-states along the catalytic cycle. Because of the complexity of the signals, only a limited quantity of identified peaks have been assigned so far, also because of the difficulty of a direct comparison with theoretical calculations. In the present work, we critically reconsider the comparison between differential vibrational spectroscopy and theoretical calculations performed on the structural models of the photosystem II active site and an inorganic structural mimic. Several factors are currently limiting the reliability of a quantitative comparison, such as intrinsic errors associated to theoretical methods, and most of all, the uncertainty attributed to the lack of knowledge about the localization of the underlying structural changes. Critical points in this comparison are extensively discussed. Comparing several computational data of differential S2 /S1 infrared spectroscopy, we have identified weak and strong points in their interpretation when compared with experimental spectra.

Evolution from S3 to S4 States of the Oxygen-Evolving Complex in Photosystem†II Monitored by Quantum Mechanics/Molecular Mechanics (QM/MM) Dynamics.

Water oxidation in the early steps of natural photosynthesis is fulfilled by photosystem II, which is a protein complex embedded in the thylakoid membrane inside chloroplasts. The water oxidation reaction occurs in the catalytic core of photosystem II, which consists of a Mn4Ca metal cluster, at which, after the accumulation of four oxidising equivalents through five steps (S0-S4) of the Kok-Joliot cycle, two water molecules are split into electrons, protons, and molecular oxygen. In recent years, by combining experimental and theoretical approaches, new insights have been achieved into the structural and electronic properties of different steps of the catalytic cycle. Nevertheless, the exact catalytic mechanism, especially concerning the final stages of the cycle, remains elusive and greatly debated. Herein, by means of quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations, from the structural, electronic, and magnetic points of view, the S3 state before and upon oxidation has been characterised. In contrast with the S2 state, the oxidation of the S3 state is not followed by a spontaneous proton-coupled electron-transfer event. Nevertheless, upon modelling the reduction of the tyrosine residue in photosystem II (TyrZ ) and the protonation of Asp61, spontaneous proton transfer occurs, leading to the deprotonation of an oxygen atom bound to Mn1; thus making it available for O-O bond formation.

Vibrational fingerprints of the Mn4CaO5 cluster in Photosystem II by mixed quantum-classical molecular dynamics.

A detailed knowledge of the structures of the catalytic steps along the Kok-Joliot cycle of Photosystem II may help to understand the strategies adopted by this unique enzyme to achieve water oxidation. Vibrational spectroscopy has probed in the last decades the intermediate states of the catalytic cycle, although the interpretation of the data turned out to be often problematic. In the present work we use QM/MM molecular dynamics on the S2 state to obtain the vibrational density of states at room temperature. To help the interpretation of the computational and experimental data we propose a decomposition of the Mn4CaO5 moiety into five separate parts, composed by "diamond" motifs involving four atoms. The spectral signatures arising from this analysis can be easily interpreted to assign experimentally known bands to specific molecular motions. In particular, we focused in the low frequency region of the vibrational spectrum of the S2 state. We can therefore identify the observed bands around 600-620cm(-1) as characteristic for the stretching vibrations involving Mn1-O1-Mn2 or Mn3-O5 moieties.

Mechanism of Water Delivery to the Active Site of Photosystem II along the S(2) to S(3) Transition.

The two water molecules serving as substrate for the oxygen evolution in Photosystem II are already bound in the S2 state of the Kok-Joliot's cycle. Nevertheless, an additional water molecule is supposed to bind the cluster during the transition between the S2 and S3 states, which has been recently revealed to have the Mn4CaO5 catalytic cluster arranged in an open cubane fashion. In this Letter, by means of ab initio calculations, we investigated the possible pathways for the binding of the upcoming water molecule. Upon the four different possibilities checked in our calculations, the binding of the crystallographic water molecule, originally located nearby the Cl(-) binding site, showed the lowest activation energy barrier. Our findings therefore support the view in which the W2 hydroxyl group and the O5 oxygen act as substrates for the oxygen evolution. Within this framework the role of the open and closed Mn4CaO5 conformers is clarified as well as the exact mechanistic events occurring along the S2 to S3 transition.

Reorganization of substrate waters between the closed and open cubane conformers during the S2 to S3 transition in the oxygen evolving complex.

A crucial step in the mechanism for oxygen evolution in the Photosystem II complex resides in the transition from the S2 state to the S3 state of Kok­Joliot's cycle, in which an additional water molecule binds to the cluster. On the basis of computational chemistry calculations on Photosystem II models, we propose a reorganization mechanism involving a hydroxyl (W2) and a µ2-oxo bridge (O5) that is able to link the closed cubane S2B intermediate conformer to the S3 open cubane structure. This mechanism can reconcile the apparent conflict between recently reported water exchange and electron paramagnetic resonance experiments, and theoretical studies.