A finely balanced order-disorder equilibrium sculpts the folding-binding landscape of an antibiotic sequestering protein.

TipA, a MerR family transcription factor from Streptomyces lividans, promotes antibiotic resistance by sequestering broad-spectrum thiopeptide-based antibiotics, thus counteracting their inhibitory effect on ribosomes. TipAS, a minimal binding motif which is expressed as an isoform of TipA, harbors a partially disordered N-terminal subdomain that folds upon binding multiple antibiotics. The extent and nature of the underlying molecular heterogeneity in TipAS that shapes its promiscuous folding-function landscape is an open question and is critical for understanding antibiotic-sequestration mechanisms. Here, combining equilibrium and time-resolved experiments, statistical modeling, and simulations, we show that the TipAS native ensemble exhibits a pre-equilibrium between binding-incompetent and binding-competent substates, with the fully folded state appearing only as an excited state under physiological conditions. The binding-competent state characterized by a partially structured N-terminal subdomain loses structure progressively in the physiological range of temperatures, swells on temperature increase, and displays slow conformational exchange across multiple conformations. Binding to the bactericidal antibiotic thiostrepton follows a combination of induced-fit and conformational-selection-like mechanisms, via partial binding and concomitant stabilization of the binding-competent substate. These ensemble features are evolutionarily conserved across orthologs from select bacteria that infect humans, underscoring the functional role of partial disorder in the native ensemble of antibiotic-sequestering proteins belonging to the MerR family.

Supramolecular citrate poly allylamine hydrochloride nanoparticles for citrate delivery and calcium oxalate nanocrystal dissolution.

HYPOTHESIS: Renal calculi (kidney stones) are mainly made by calcium oxalate and can cause different complications including malfunction of the kidney. The most important urinary stone inhibitors are citrate molecules. Unfortunately, the amount of citrate reaching the kidney after oral ingestion is low. We hypothesized that nanoparticles of polyallylamine hydrochloride (CIT-PAH) carrying citrate ions could simultaneously deliver citrates while PAH would complex oxalate triggering dissolution and removal of CaOx nanocrystals. EXPERIMENTS: We successfully prepared nanoparticles of citrate ions with polyallylamine hydrochloride (CIT-PAH), PAH with oxalate (OX-PAH) and characterize them by Small Angle X ray Scattering (SAXS), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and NMR. Dissolution of CaOx nanocrystals in presence of CIT-PAH have been followed with Wide Angle Xray Scattering (WAXS), DLS and Confocal Raman Microscopy. Raman spectroscopy was used to study the dissolution of crystals in synthetic urine samples. The release of citrate from CIT-PAH was followed by diffusion NMR. Molecular dynamics (MD) simulations were carried out to study the interaction of CIT and OX ions with PAH. FINDINGS: CIT-PAH nanoparticles dissolves CaOx nanocrystals as shown by NMR, DLS, TEM and WAXS in water and by Raman spectroscopy in artificial human urine. WAXS and Raman show that the crystal structure of CaOx disappears in the presence of CIT-PAH. DLS shows that the time required for CaOX dissolution will depend on the concentration of CIT-PAH NPs. NMR proves that citrate ions are released from the CIT PAH NPs during CaOX dissolution, MD simulations showed that oxalates exhibit a stronger interaction for PAH than citrate, explaining the removal of oxalate ions and replacement of the citrate in the polymer nanoparticles.

The role of the macroalgae Ulva lactuca on the cellular effects of neodymium and mercury in the mussel Mytilus galloprovincialis.

Rare earth elements (REEs) are increasingly being studied mainly due to their economic importance and wide range of applications, but also for their rising environmental concentrations and potential environmental and ecotoxicological impacts. Among REEs, neodymium (Nd) is widely used in lasers, glass additives, and magnets. Currently, NdFeB-based permanent magnets are the most significant components of electronic devices and Nd is used because of its magnetic properties. In addition to REEs, part of the environmental pollution related to electrical and electronic equipment, fluorescent lamps and batteries also comes from mercury (Hg). Since both elements persist in ecosystems and are continuously accumulated by marine organisms, a promising approach for water decontamination has emerged. Through a process known as sorption, live marine macroalgae can be used, especially Ulva lactuca, to accumulate potential toxic elements from the water. Therefore, the present study aimed to evaluate the cellular toxicity of Nd and Hg in Mytilus galloprovincialis, comparing the biochemical effects induced by these elements in the presence or absence of the macroalgae U. lactuca. The results confirmed that Hg was more toxic to mussels than Nd, but also showed the good capability of U. lactuca in preventing the onset of cellular disturbance and homeostasis disruption in M. galloprovincialis by reducing bioavailable Hg levels. Overall, the biochemical parameters evaluated related to metabolism, antioxidant and biotransformation defences, redox balance, and cellular damage, showed that algae could prevent biological effects in mussels exposed to Hg compared to those exposed to Nd. This study contributes to the advancement of knowledge in this field, namely the understanding of the impacts of different elements on bivalves and the crucial role of algae in the protection of other aquatic organisms.

Can exposure to Gymnodinium catenatum toxic blooms influence the impacts induced by Neodymium in Mytilus galloprovincialis mussels? What doesn't kill can make them stronger?

The presence in marine shellfish of toxins and pollutants like rare earth elements (REEs) poses a major threat to human well-being, coastal ecosystems, and marine life. Among the REEs, neodymium (Nd) stands out as a widely utilized element and is projected to be among the top five critical elements by 2025. Gymnodinum catenatum is a phytoplankton species commonly associated with the contamination of bivalves with paralytic shellfish toxins. This study evaluated the biological effects of Nd on the mussel species Mytilus galloprovincialis when exposed to G. catenatum cells for fourteen days, followed by a recovery period in uncontaminated seawater for another fourteen days. After co-exposure, mussels showed similar toxin accumulation in the Nd and G. catenatum treatment in comparison with the G. catenatum treatment alone. Increased metabolism and enzymatic defenses were observed in organisms exposed to G. catenatum cells, while Nd inhibited enzyme activity and caused cellular damage. Overall, this study revealed that the combined presence of G. catenatum cells and Nd, produced positive synergistic effects on M. galloprovincialis biochemical responses compared to G. catenatum alone, indicating that organisms' performance may be significantly modulated by the presence of multiple co-occurring stressors, such those related to chemical pollution and harmful algal blooms. ENVIRONMENTAL IMPLICATIONS: Neodymium (Nd) is widely used in green technologies like wind turbines, and this element's potential threats to aquatic environments are almost unknown, especially when co-occurring with other environmental factors such as blooms of toxic algae. This study revealed the cellular impacts induced by Nd in the bioindicator species Mytilus galloprovincialis but further demonstrated that the combination of both stressors can generate a positive defense response in mussels. The present findings also demonstrated that the impacts caused by Nd lasted even after a recovery period while a previous exposure to the toxins generated a faster biochemical improvement by the mussels.

Unveiling the Cleavage Mechanism of an RNA Model Compound on the whole pH Scale: Computations Meet Experiments in the Determination of Reaction Rates.

The knowledge of the mechanism of reactions occurring in solution is a primary research line both in the context of theoretical-computational chemistry and in the field of organic and bio-organic chemistry. Given the importance of the hydrolysis of nucleic acids in life-related phenomena, here we present a combined experimental and computational study on the cleavage of an RNA model compound. This phosphodiester features a cleavage rate strictly dependent on the pH with three different dependence domains. Such experimental evidence, highlighted by an in-depth kinetic investigation, unequivocally suggests a change in the reaction mechanism along the pH scale. In order to interpret the data and to explain the experimental behavior, we have applied a theoretical-computational procedure, involving a hybrid quantum/classical approach, able to model chemical reactions in complex environments, i. e. in solution. This study turns out to quantitatively reproduce the experimental data with accuracy and, in addition, provides useful mechanistic insight into the transesterification process of the investigated compound. The study indicates that the cleavage can occur through an A N D N ${A_N D_N }$ , an A N + D N ${A_N + D_N }$ , and a D N A N ${D_N A_N }$ mechanism depending on the pH values.

A Theoretical-Computational Study of Phosphodiester Bond Cleavage Kinetics as a Function of the Temperature.

The hydrolysis of the phosphodiester bond is an important chemical reaction involved in several biological processes. Here, we study the cleavage of this bond by means of a theoretical-computational method in a model system, the dineopentyl phosphate. By such an approach, we reconstructed the kinetics and related thermodynamics of this chemical reaction along an isochore. In particular, we evaluated the kinetic constants of all the reaction steps within a wide range of temperatures, mostly corresponding to conditions where no experimental measures are available due to the extremely slow kinetics. Our results, in good agreement with the experimental data, show the robustness of our theoretical-computational methodology which can be easily extended to more complex systems.

Cellular effects of microplastics are influenced by their dimension: Mechanistic relationships and integrated criteria for particles definition.

The definition of microplastics (MPs) is nowadays too generic from a biological perspective, since different characteristics of these particles might influence their effects. To provide experimental evidence that size is an important factor to be considered, Mediterranean mussels Mytilus galloprovincialis were exposed to five size classes of polyethylene fragments (PE-MPs, 20-50 µm, 50-100 µm, 100-250 µm, 250-500 µm, 500-1000 µm). After 10 days of exposure, MPs ingestion and mechanistic relationships between particles size and cellular effects were analysed through a wide panel of biological alterations, including immune system responses, cholinergic function, antioxidant system, lipid metabolism and peroxidation. Results were further elaborated through a Weight of Evidence approach, summarizing the overall biological significance of obtained results in a hazard index based on the number and magnitude of variations and their toxicological relevance. PE-MPs 500-1000 µm were identified as the less biologically reactive size class due to the limited ingestion of particles coupled with the lack of biological effects, followed by PE-MPs 250-500 µm, which slightly altered the cholinergic function and lysosomal membranes. Conversely, PE-MPs smaller than 250 µm provoked a more consistent onset of biological alterations in terms of immune system composition and functioning, redox homeostasis, and lipid metabolism. The overall findings of this study highlight the importance of considering the size of particles for monitoring and risk assessment of MPs, introducing a more integrated evaluation of plastic pollution that, beside particles concentration, should adequately weigh those characteristics triggering the onset of biological effects.

The EU Interreg Project "GEREMIA" on waste management for the improvement of port waters: results on monitoring the health status of fish as bioindicator.

Highly anthropized areas as ports represent complex scenarios that require accurate monitoring plans aimed to address the environmental status. In this context, the activities of the EU Interreg Project "GEstione dei REflui per il MIglioramento delle Acque portuali (GEREMIA)" were focused on comparing sites differently affected by human presence, as the Port of Genoa and the natural area of the S'Ena Arrubia fishpond: a panel of analyses was carried out on Mugilidae fish sampled in these two areas, aimed to address trace metal accumulation in the liver, gills, and muscle, as well as cytochrome P450 (CYP450) induction in liver and biliary polycyclic aromatic hydrocarbon (PAH) metabolites, and histopathological alterations in the liver and gills. Chemical analyses in the liver, gills, and muscle of specimens collected in the port area showed an overall higher degree of trace metal contamination compared to the natural fishpond, and similar results were obtained in terms of CYP450 induction and biliary PAH metabolites, suggesting a higher exposure to organic compounds. In addition, histopathological analyses revealed a significant alteration and then a loss of functionality of liver and gill tissue in individuals from the port. Overall, this study describes the complex environmental pollution scenario in the Port of Genoa, confirming the importance of using multidisciplinary approaches and different types of analyses to address both the presence and the effects of contaminants in marine environments.

Molecular Modeling of the Deamidation Reaction in Solution: A Theoretical-Computational Study.

In this work, a theoretical-computational method is applied to study the deamidation reaction, a critical post-translational modification in proteins, using a simple model molecule in solution. The method allows one to comprehensively address the environmental effect, thereby enabling one to accurately derive the kinetic rate constants for the three main steps of the deamidation process. The results presented, in rather good agreement with the available experimental data, underline the necessity for a rigorous treatment of environmental factors and a precise kinetic model to correctly assess the overall kinetics of the deamidation reaction.

A study of cyanidin/alginate complexation: Influence of pH in assembly and chiral properties.

Cyanidin 3-O-glucoside (CND) is a frequently-used anthocyanin that has excellent antioxidant properties but a limited bioavailability in bloodstream. Complexation of CND with alginate can improve its therapeutic outcome. Here we have studied the complexation of CND with alginate under a range of pH values from 2.5 to 5. CND is positively charged at low pH, and becomes neutral, and then negatively charged as pH increases. CND/alginate complexation was studied by dynamic light scattering, transmission electron microscopy, small angle X-ray scattering, STEM, UV-Vis spectroscopy and circular dichroism (CD). CND/alginate complexes at pH 4.0 and 5.0 form chiral fibres with a fractal structure. At these pH values, CD spectra show very intense bands, which are inverted compared with free CND. Complexation at lower pH results in disordered polymer structures and CD spectra show the same features as for CND in solution. Molecular dynamics simulations suggest the formation of parallel CND dimers through complexation with alginate at pH 3.0, while at pH 4.0 CND dimers form in a cross like arrangement.

Modelling Complex Bimolecular Reactions in a Condensed Phase: The Case of Phosphodiester Hydrolysis.

(1) Background: the theoretical modelling of reactions occurring in liquid phase is a research line of primary importance both in theoretical-computational chemistry and in the context of organic and biological chemistry. Here we present the modelling of the kinetics of the hydroxide-promoted hydrolysis of phosphoric diesters. (2) Method: the theoretical-computational procedure involves a hybrid quantum/classical approach based on the perturbed matrix method (PMM) in conjunction with molecular mechanics. (3) Results: the presented study reproduces the experimental data both in the rate constants and in the mechanistic aspects (C-O bond vs. O-P bond reactivity). The study suggests that the basic hydrolysis of phosphodiesters occurs through a concerted ANDN mechanism, with no formation of penta-coordinated species as reaction intermediates. (4) Conclusions: the presented approach, despite the approximations, is potentially applicable to a large number of bimolecular transformations in solution and therefore leads the way to a fast and general method to predict the rate constants and reactivities/selectivities in complex environments.

Effect of Salts on the Conformational Dynamics of the Cytochrome P450 OleP.

Cytochrome P450 OleP catalytic activity is strongly influenced by its structural dynamic conformational behavior. Here, we combine equilibrium-binding experiments with all-atom molecular dynamics simulations to clarify how different environments affect OleP conformational equilibrium between the open and the closed-catalytic competent-forms. Our data clearly show that at high-ionic strength conditions, the closed form is favored, and, very interestingly, different mechanisms, depending on the chemistry of the cations, can be used to rationalize such an effect.

PyMM: An Open-Source Python Program for QM/MM Simulations Based on the Perturbed Matrix Method.

Quantum mechanical/molecular mechanics (QM/MM) methods are important tools in molecular modeling as they are able to couple an extended phase space sampling with an accurate description of the electronic properties of the system. Here, we describe a Python software package, called PyMM, which has been developed to apply a QM/MM approach, the perturbed matrix method, in a simple and efficient way. PyMM requires a classical atomic trajectory of the whole system and a set of unperturbed electronic properties of the ground and electronic excited states. The software output includes a set of the most common perturbed properties, such as the electronic excitation energies and the transitions dipole moments, as well as the eigenvectors describing the perturbed electronic states, which can be then used to estimate whatever electronic property. The software is composed of a simple and complete command-line interface, a set of internal input validation, and three main analyses focusing on (i) the perturbed eigenvector behavior, (ii) the calculation of the electronic absorption spectrum, and (iii) the estimation of the free energy differences along a reaction coordinate.

Modeling Charge Transfer Reactions by Hopping between Electronic Ground State Minima: Application to Hole Transfer between DNA Bases.

In this paper, we extend the previously described general model for charge transfer reactions, introducing specific changes to treat the hopping between energy minima of the electronic ground state (i.e., transitions between the corresponding vibrational ground states). We applied the theoretical-computational model to the charge transfer reactions in DNA molecules which still represent a challenge for a rational full understanding of their mechanism. Results show that the presented model can provide a valid, relatively simple, approach to quantitatively study such reactions shedding light on several important aspects of the reaction mechanism.

Complexation and organization of doxorubicin on polystyrene sulfonate chains: impacts on doxorubicin dimerization and quenching.

Anthracycline doxorubicin hydrochloride (DX) is a positively charged fluorescent drug, which in water self-associates into non-fluorescent antiparallel dimers upon increasing concentration and/or ionic strength. The positive charge of DX allows for complexation with negatively charged polymers and drug carriers. The fluorescence of DX following complexation with polyanion polystyrene sulfonate (PSS) is studied here. The fluorescence emission of DX decreases in the presence of PSS, being almost completely quenched when the ratio (R) of PSS monomers-to-DX molecules is larger than 10. Increasing R values over 30 results in a progressive recovery of fluorescence. The circular dichroism of PSS-DX complexes shows inverted characteristic bands of DX dimers suggesting the presence of parallel dimers at a concentration of DX below dimerization in water. Molecular dynamics studies corroborate a preferential orientation of DX into parallel dimers when interacting with PSS and show that DX molecules interact with a binding pocket of PSS monomers rather than with one single monomer. Increasing the ionic strength results in a recovery of fluorescence without an apparent release of DX from the PSS-DX complex as shown by DOSY NMR. PSS acts as a template for concentrating DX, triggering dimerisation and orienting DX molecules with their charged groups facing the negatively charged PSS monomers.

Microplastics in seawater and marine organisms: Site-specific variations over two-year study in Giglio Island (North Tyrrhenian Sea).

Geographical and temporal differences of microplastic occurrence were documented in water and fish collected in 2017 and 2019 from the Giglio Island (North Tyrrhenian Sea) close to the area where the Costa Concordia sank in January 2012. Results on water samples showed a site-dependent difference, suggesting the role of surface current dynamics in the microplastic local distribution, while tested Neuston nets (200 µm and 330 µm mesh size) did not influence microplastic retention efficiency. Fish exhibited in 2019 a higher frequency of specimens positive to microplastic ingestion with respect to 2017, with an occurrence higher than those typically observed in other Mediterranean areas. Both in water and fish, fragments were the dominating shape, polypropylene and polyethylene were the prevalent polymers, without particular difference between sites and years. This study highlights the importance of applying microplastic investigation in biotic and abiotic matrices for an effective monitoring of this pollution in the marine environment.

Rationalizing Sequence and Conformational Effects on the Guanine Oxidation in Different DNA Conformations.

The effect of the environment on the guanine redox potential is studied by means of a theoretical-computational approach. Our data, in agreement with previous experimental findings, clearly show that the presence of consecutive guanine bases in both single- and double-stranded DNA oligomers lowers their reduction potential. Such an effect is even more marked when a G-rich quadruplex is considered, where the oxidized form of guanine is particularly stabilized. To the best of our knowledge, this is the first computational study reporting on a quantitative estimate of the dependence of the guanine redox potential on sequence and conformational effects in complex DNA molecules, ranging from single-stranded DNA to G-quadruplex.

Absorption behavior of doxorubicin hydrochloride in visible region in different environments: a combined experimental and computational study.

The experimental absorption measurements in the interval 350-600 nm (Vis), molecular dynamics simulations, quantum-mechanics calculations and an advanced molecular treatment of simulation data are here combined to provide a complete picture of the absorption behavior in the visible portion of the electromagnetic spectrum of the doxorubicin hydrochloride (DX) molecule in different environments. By such an approach, we have shown that it is possible to characterize the effect of the environment on the DX absorption behavior - including the vibronic contributions - as well as to interpret such differences in terms of molecular electronic excited states, which are found to be strongly influenced by the environment.

Marine heatwaves hamper neuro-immune and oxidative tolerance toward carbamazepine in Mytilus galloprovincialis.

The increased frequency and intensity of short-term extreme warming phenomena have been associated to harsh biological and ecosystem outcomes (i.e., mass mortalities in marine organisms). Marine heatwaves (MHWs), occurring when seasonal temperature threshold is exceeded for at least 5 consecutive days, may reduce the tolerance of coastal species toward additional pressures, but interactions between such multiple stressors are virtually unexplored. The present study aimed to characterize in Mytilus galloprovincialis the influence of a simulated MHW scenario on the toxicological effects of the pharmaceutical carbamazepine (CBZ), ubiquitously detected in the marine environment and chosen as model compound for this relevant class of emerging contaminants. The bioaccumulation of CBZ and responsiveness of various biological parameters, including immune system, antioxidant status, lipid metabolism and cellular integrity, were analyzed in exposed mussels both during and after the end of the heatwave. MHW appeared to strongly modulate accumulation of CBZ, paralleled by weakened immunocompetence and onset of oxidative disturbance that finally evolved to cellular damages and lipid metabolism disorders. Elaboration of the overall results through a quantitative Weight of Evidence model, revealed the highest hazard in organisms exposed to both the stressors 10 days after the end of the heatwave, suggesting that MHWs could leave a footprint on the capability of mussels to counteract CBZ toxicity, thus affecting their vulnerability and predisposition to adverse effects toward multiple stressors.

Theoretical Modeling of Redox Potentials of Biomolecules.

The estimation of the redox potentials of biologically relevant systems by means of theoretical-computational approaches still represents a challenge. In fact, the size of these systems typically does not allow a full quantum-mechanical treatment needed to describe electron loss/gain in such a complex environment, where the redox process takes place. Therefore, a number of different theoretical strategies have been developed so far to make the calculation of the redox free energy feasible with current computational resources. In this review, we provide a survey of such theoretical-computational approaches used in this context, highlighting their physical principles and discussing their advantages and limitations. Several examples of these approaches applied to the estimation of the redox potentials of both proteins and nucleic acids are described and critically discussed. Finally, general considerations on the most promising strategies are reported.