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We produced a neuroglobin variant, namely, Ngb CDless, with the excised CDloop- and D-helix, directly joining the C- and E-helices. The CDless variant retained bis-His hexacoordination, and we investigated the role of the CDloop-D-helix unit in controlling the CO binding and structural dynamics by an integrative approach based on X-ray crystallography, rapid mixing, laser flash photolysis, resonance Raman spectroscopy, and molecular dynamics simulations. Rapid mixing and laser flash photolysis showed that ligand affinity was unchanged with respect to the wild-type protein, albeit with increased on and off constants for rate-limiting heme iron hexacoordination by the distal His64. Accordingly, resonance Raman spectroscopy highlighted a more open distal pocket in the CO complex that, in agreement with MD simulations, likely involves His64 swinging inward and outward of the distal heme pocket. Ngb CDless displays a more rigid overall structure with respect to the wild type, abolishing the structural dynamics of the CDloop-D-helix hypothesized to mediate its signaling role, and it retains ligand binding control by distal His64. In conclusion, this mutant may represent a tool to investigate the involvement of CDloop-D-helix in neuroprotective signaling in a cellular or animal model.

Coproporphyrin ferrochelatases (CpfCs) are enzymes catalyzing the penultimate step in the coproporphyrin-dependent (CPD) heme biosynthesis pathway, which is mainly utilized by monoderm bacteria. Ferrochelatases insert ferrous iron into a porphyrin macrocycle and have been studied for many decades, nevertheless many mechanistic questions remain unanswered to date. Especially CpfCs, which are found in the CPD pathway, are currently in the spotlight of research. This pathway was identified in 2015 and revealed that the correct substrate for these ferrochelatases is coproporphyrin III (cpIII) instead of protoporphyrin IX, as believed prior the discovery of the CPD pathway. The chemistry of cpIII, which has four propionates, differs significantly from protoporphyrin IX, which features two propionate and two vinyl groups. These findings let us to thoroughly describe the physiological cpIII-ferrochelatase complex in solution and in the crystal phase. Here, we present the first crystallographic structure of the CpfC from the representative monoderm pathogen Listeria monocytogenes bound to its physiological substrate, cpIII, together with the in-solution data obtained by resonance Raman and UV-vis spectroscopy, for wild-type ferrochelatase and variants, analyzing propionate interactions. The results allow us to evaluate the porphyrin distortion and provide an in-depth characterization of the catalytically-relevant binding mode of cpIII prior to iron insertion. Our findings are discussed in the light of the observed structural restraints and necessities for this porphyrin-enzyme complex to catalyze the iron insertion process. Knowledge about this initial situation is essential for understanding the preconditions for iron insertion in CpfCs and builds the basis for future studies.

Coproporpyhrin III is the substrate of coproporphyrin ferrochelatases (CpfCs). These enzymes catalyse the insertion of ferrous iron into the porphyrin ring. This is the penultimate step within the coproporphyrin-dependent haeme biosynthesis pathway. This pathway was discovered in 2015 and is mainly utilised by monoderm bacteria. Prior to this discovery, monoderm bacteria were believed to utilise the protoporphyrin-dependent pathway, analogously to diderm bacteria, where the substrate for the respective ferrochelatase is protoporphyrin IX, which has two propionate groups at positions 6 and 7 and two vinyl groups at positions 2 and 4. In this work, we describe for the first time the interactions of the four-propionate substrate, coproporphyrin III, and the four-propionate product, iron coproporphyrin III (coproheme), with the CpfC from Listeria monocytogenes and pin down differences with respect to the protoporphyrin IX and haeme b complexes in the wild-type (WT) enzyme. We further created seven LmCpfC variants aiming at altering substrate and product coordination. The WT enzyme and all the variants were comparatively studied by spectroscopic, thermodynamic and kinetic means to investigate in detail the H-bonding interactions, which govern complex stability and substrate specificity. We identified a tyrosine residue (Y124 in LmCpfC), coordinating the propionate at position 2, which is conserved in monoderm CpfCs, to be highly important for binding and stabilisation. Importantly, we also describe a tyrosine-serine-threonine triad, which coordinates the propionate at position 4. The study of the triad variants indicates structural differences between the coproporphyrin III and the coproheme complexes. ENZYME: EC 4.99.1.9.

Hydrophobicity/hydrophilicity of aqueous interfaces at the molecular level results from a subtle balance in the water-water and water-surface interactions. This is characterized here via density functional theory-molecular dynamics (DFT-MD) coupled with vibrational sum frequency generation (SFG) and THz-IR absorption spectroscopies. We show that water at the interface with a series of weakly interacting materials is organized into a two-dimensional hydrogen-bonded network (2D-HB-network), which is also found above some macroscopically hydrophilic silica and alumina surfaces. These results are rationalized through a descriptor that measures the number of "vertical" and "horizontal" hydrogen bonds formed by interfacial water, quantifying the competition between water-surface and water-water interactions. The 2D-HB-network is directly revealed by THz-IR absorption spectroscopy, while the competition of water-water and water-surface interactions is quantified from SFG markers. The combination of SFG and THz-IR spectroscopies is thus found to be a compelling tool to characterize the finest details of molecular hydrophobicity at aqueous interfaces.

Nitrobindins (Nbs) are all-β-barrel heme-proteins present in prokaryotes and eukaryotes. Although the physiological role(s) of Nbs are still unclear, it has been postulated that they are involved in the NO/O2 metabolism, which is particularly relevant in fishes for the oxygen supply. Here, the reactivity of ferrous Danio rerio Nb (Dr-Nb(II)) towards NO has been investigated from the spectroscopic and kinetic viewpoints and compared with those of Mycobacterium tuberculosis Nb, Arabidopsis thaliana Nb, Homo sapiens Nb, and Equus ferus caballus myoglobin. Between pH 5.5 and 9.1 at 22.0 °C, Dr-Nb(II) nitrosylation is a monophasic process; values of the second-order rate constant for Dr-Nb(II) nitrosylation and of the first-order rate constant for Dr-Nb(II)-NO denitrosylation are pH-independent ranging between 1.6 × 106 M-1 s-1 and 2.3 × 106 M-1 s-1 and between 5.3 × 10-2 s-1 and 8.2 × 10-2 s-1, respectively. Interestingly, both UV-Vis and EPR spectroscopies indicate that the heme-Fe(II) atom of Dr-Nb(II)-NO is five-coordinated. Kinetics of Dr-Nb(II) nitrosylation may reflect the ligand accessibility to the metal center, which is likely impaired by the crowded network of water molecules which shields the heme pocket from the bulk solvent. On the other hand, kinetics of Dr-Nb(II)-NO denitrosylation may reflect an easy pathway for the ligand escape into the outer solvent.

International audience; Water, being the universal solvent, acts as a competing agent in fundamental processes, such as folding, aggregation or biomolecular recognition. A molecular understanding of hydrophobic hydration is of central importance to understanding the subtle free energy differences, which dictate function. Ab initio and classical molecular dynamics simulations yield two distinct hydration water populations in the hydration shell of solvated tert-butanol noted as “HB-wrap” and “HB-hydration2bulk”. The experimentally observed hydration water spectrum can be dissected into two modes, centered at 164 and 195 cm–1. By comparison to the simulations, these two bands are attributed to the “HB-wrap” and “HB-hydration2bulk” populations, respectively. We derive a quantitative correlation between the population in each of these two local water coordination motifs and the temperature dependence of the solvation entropy. The crossover from entropy to enthalpy dominated solvation at elevated temperatures, as predicted by theory and observed experimentally, can be rationalized in terms of the distinct temperature stability and thermodynamic signatures of “HB-wrap” and “HB-hydration2bulk”.

Arsenic is one of the most prevalent toxic elements in the environment, and its toxicity affects every organism. Arsenic resistance has mainly been observed in microorganisms, and, in bacteria, it has been associated with the presence of the Ars operon. In

Hydration free energies are dictated by a subtle balance of hydrophobic and hydrophilic interactions. which is crucial for many biological processes and technological applications, such as protein folding and molecular recognition. Whereas so far the overall entropy and enthalpy are experimentally determined based on equilibrium measurements using a calorimeter, we present here a pure spectroscopic access to these important observables, which give direct access to the underlying molecular mechanism that determines these driving forces. Using THz calorimetry the contributions due to cavity formation and hydrophilic interactions can be traced back to changes in the intermolecular hydrogen bond stretching region around 150-200 cm−1 and spectroscopic changes due to strong solute-water interactions in the frequency range of the librational modes, i.e. between 540 and 600 cm−1. Thus, we are able to link the thermodynamic model of the Lum-Chandler-Weeks theory, which was a pure ”Gedankenexperiment”, directly to experimental observables. We show that alcohol hydration can be described by a sum of a free energy cost of forming and wrapping a cavity around the solute (which is entropic for small alcohols) and an enthalpic gain due to the hydrogen bonds formed between the alcohol OH group and bound water molecules around it. In the future, our approach will allow to quantify entropic cost and enthalpic gain not only in equilibrium but also in non-equilibrium processes.

Hydration free energies are dictated by a subtle balance of hydrophobic and hydrophilic interactions. We present here a spectroscopic approach, which gives direct access to the two main contributions: Using THz-spectroscopy to probe the frequency range of the intermolecular stretch (150-200 cm

Coproheme decarboxylases (ChdCs) are utilized by monoderm bacteria to produce heme b by a stepwise oxidative decarboxylation of the 2- and 4-propionate groups of iron coproporphyrin III (coproheme) to vinyl groups. This work compares the effect of hemin reconstitution versus the hydrogen peroxide-mediated conversion of coproheme to heme b in the actinobacterial ChdC from Corynebacterium diphtheriae (CdChdC) and selected variants. Both ferric and ferrous forms of wild-type (WT) CdChdC and its H118A, H118F, and A207E variants were characterized by resonance Raman and UV-vis spectroscopies. The heme b ligand assumes the same conformation in the WT active site for both the reconstituted and H

Riparian ecosystems, in long-time developed regions, are among the most heavily impacted by human activities; therefore, the distribution of tree riparian species, such as

Significance For centuries the double layer at the solid/electrolyte interface has been a central concept in electrochemistry. Today, it is still crucial for virtually all renewable energy storage and conversion technologies. Here, the double-layer formation is probed by THz spectroscopy with ultrabright synchrotron light as a source. Our results capture the molecular details of double-layer formation at positively/negatively charged Au electrodes for an NaCl electrolyte. We reveal a contrasting response applying positive versus negative bias, which is dictated by the interfacial water network and rationalized by accompanying molecular dynamics simulations and electronic-structure calculations. While Na+ is directly attracted toward the negatively charged electrode, stripping of the Cl− hydration shell is observed only at larger potential values., The double layer at the solid/electrolyte interface is a key concept in electrochemistry. Here, we present an experimental study combined with simulations, which provides a molecular picture of the double-layer formation under applied voltage. By THz spectroscopy we are able to follow the stripping away of the cation/anion hydration shells for an NaCl electrolyte at the Au surface when decreasing/increasing the bias potential. While Na+ is attracted toward the electrode at the smallest applied negative potentials, stripping of the Cl− hydration shell is observed only at higher potential values. These phenomena are directly measured by THz spectroscopy with ultrabright synchrotron light as a source and rationalized by accompanying molecular dynamics simulations and electronic-structure calculations.

Pollution affects most of the urban and forest environments at different levels causing well-known effects on human and plant health. The influence that pollutants exert on plant-associated microbiota might direct plant health and, in some cases, also the removal of pollutants by plants. With the advent of nanotechnologies, an increasing amount of engineered nanoparticles are being introduced into the environment, and consequently, their impact on plant-associated microorganisms needs to be investigated. In this context, silver nanoparticles (Ag-NPs) were experimentally supplied at leaf and root level of poplar plants to assess Ag-NPs effects on plant microbiota. Leaf Ag-NP treatment increased bacteria and fungi evenness and determined a significant reduction in both microbial groups, while root Ag-NP treatment reduced the bacterial and fungal biodiversity. Bioinformatics functional analysis showed that Ag-NP treatment reduced the aerobic and stimulated facultative anaerobic and oxidative stress-tolerant bacteria. Our study offers new insights into the effects of Ag-NPs on both phyllosphere and rhizosphere poplar-associated microbiota and may represent a first attempt to understand the behavior of microbial communities of a tree species growing in a polluted environment.

Simple Summary Chronic enteropathies are common gastrointestinal diseases in domestic dogs characterised by long-term duration, often impairing quality of life both for pets and owners. It has been demonstrated that the gut microbial community plays a central role in defining the host health status. Indeed, among a variety of biological functions, gut microbiota are involved in the metabolism of nutrients, in training the immune system and in preventing the gastrointestinal ecosystem from being colonised by pathogens. In chronic intestinal diseases, the equilibrium of the gut microbial population is largely impaired, as a consequence of both disease and therapy (e.g., antibiotic treatment). Faecal microbiota transplantation has the aim to restore a balanced microbial population in the patient by simply implanting a healthy gut microbiota derived from a healthy donor to a diseased animal. In doing so, the eubiotic community—and the extensive network of beneficial cross-feeding interactions—are transferred to the receiver’s gut as a whole, favouring the patient to renew a healthy intestinal ecosystem. In this work, we report the encouraging results of a faecal transplantation on a 9-year-old dog suffering from chronic enteropathy for the last 3 years. After the treatment, the dog’s appetite, body weight and vitality were restored, with complete disappearance of gastrointestinal and systemic symptoms. Abstract Chronic enteropathies (CE) are gastrointestinal diseases that afflict about one in five dogs in Europe. Conventional therapeutic approaches include dietary intervention, pharmacological treatment and probiotic supplements. The patient response can be highly variable and the interventions are often not resolutive. Moreover, the therapeutic strategy is usually planned (and gradually corrected) based on the patient’s response to empirical treatment, with few indirect gut health indicators useful to drive clinicians’ decisions. The ever-diminishing cost of high-throughput sequencing (HTS) allows clinicians to directly follow and characterise the evolution of the whole gut microbial community in order to highlight possible weaknesses. In this framework, faecal microbiome transplantation (FMT) is emerging as a feasible solution to CE, based on the implant of a balanced, eubiotic microbial community from a healthy donor to a dysbiotic patient. In this study, we report the promising results of FMT carried out in a 9-year-old dog suffering from CE for the last 3 years. The patient underwent a two-cycle oral treatment of FMT and the microbiota evolution was monitored by 16S rRNA gene sequencing both prior to FMT and after the two administrations. We evaluated the variation of microbial composition by calculating three different alpha diversity indices and compared the patient and donor data to a healthy control population of 94 dogs. After FMT, the patient’s microbiome and clinical parameters gradually shifted to values similar to those observed in healthy dogs. Symptoms disappeared during a follow-up period of six months after the second FMT. We believe that this study opens the door for potential applications of FMT in clinical veterinary practice and highlights the need to improve our knowledge on this relevant topic.

The double layer at the solid/electrolyte interface is a key concept in electrochemistry. Here, we present an experimental study combined with simulations, which provides a molecular picture of the double-layer formation in operando processes. By THz spectroscopy we are able to follow the stripping off of the cation/anion hydration shells for a NaCl electrolyte at the Au surface when decreasing/increasing the bias potential. While Na+ is attracted toward the electrode already at the smallest applied negative potentials, stripping-off of the Cl- hydration shell is observed only at higher potential values. These phenomena are directly measured by in operando THz spectroscopy with ultra-bright synchrotron light as a source and rationalized by accompanying molecular-dynamics simulations and electronic-structure calculations.

Monoderm bacteria utilize coproheme decarboxylases (ChdCs) to generate heme b by a stepwise decarboxylation of two propionate groups of iron coproporphyrin III (coproheme), forming two vinyl groups. This work focuses on actinobacterial ChdC from Corynebacterium diphtheriae (CdChdC) to elucidate the hydrogen peroxide-mediated decarboxylation of coproheme via monovinyl monopropionyl deuteroheme (MMD) to heme b, with the principal aim being to understand the reorientation mechanism of MMD during turnover. Wild-type CdChdC and variants, namely H118A, H118F, and A207E, were studied by resonance Raman and ultraviolet-visible spectroscopy, mass spectrometry, and molecular dynamics simulations. As actinobacterial ChdCs use a histidine (H118) as a distal base, we studied the H118A and H118F variants to elucidate the effect of 1) the elimination of the proton acceptor and 2) steric constraints within the active site. The A207E variant mimics the proximal H-bonding network found in chlorite dismutases. This mutation potentially increases the rigidity of the proximal site and might impair the rotation of the reaction intermediate MMD. We found that both wild-type CdChdC and the variant H118A convert coproheme mainly to heme b upon titration with H2O2. Interestingly, the variant A207E mostly accumulates MMD along with small amounts of heme b, whereas H118F is unable to produce heme b and accumulates only MMD. Together with molecular dynamics simulations, the spectroscopic data provide insight into the reaction mechanism and the mode of reorientation of MMD, i.e., a rotation in the active site versus a release and rebinding.

Anthocyanins have long been suggested as having great potential in offering photoprotection to plants facing high light irradiance. Nonetheless, their effective ability in protecting the photosynthetic apparatus from supernumerary photons has been questioned by some authors, based upon the inexact belief that anthocyanins almost exclusively absorb green photons, which are poorly absorbed by chlorophylls. Here we focus on the blue light absorbing features of anthocyanins, a neglected issue in anthocyanin research. Anthocyanins effectively absorb blue photons: the absorbance of blue relative to green photons increases from tri- to mono-hydroxy B-ring substituted structures, reaching up to 50% of green photons absorption. We offer a comprehensive picture of the molecular events activated by low blue-light availability, extending our previous analysis in purple and green basil, which we suggest to be responsible for the "shade syndrome" displayed by cyanic leaves. While purple leaves display overexpression of genes promoting chlorophyll biosynthesis and light harvesting, in green leaves it is the genes involved in the stability/repair of photosystems that are largely overexpressed. As a corollary, this adds further support to the view of an effective photoprotective role of anthocyanins. We discuss the profound morpho-anatomical adjustments imposed by the epidermal anthocyanin shield, which reflect adjustments in light harvesting capacity under imposed shade and make complex the analysis of the photosynthetic performance of cyanic versus acyanic leaves.

Based upon precise terahertz (THz) measurements of the solvated amino acid glycine and accompanying ab‐initio molecular‐dynamics simulations, we show that the N‐C‐C‐O open/close mode at 315 cm−1 serves as a sensitive, label‐free probe for the local protonation of the amide group. Experimentally, we can show that this holds not only for glycine but also for diglycine and valine. The approach is more general, since the changes due to protonation result in intensity changes which can be probed by THz time domain (0–50 cm−1) as well as by precise THz‐FT spectroscopy (50–400 cm−1). A detailed analysis allows us to directly correlate the titration spectra with pK a values. This demonstrates the potential of THz spectroscopy to probe the charge state of a natural amino acid in water in a label‐free manner., Based upon precise terahertz (THz) measurements of the solvated amino acid glycine and accompanying ab‐initio molecular‐dynamics simulations, it is shown that the N‐C‐C‐O open/close mode at 315 cm−1 serves as a sensitive, label‐free probe for the local protonation of the amide group.

Significance Using a supramolecular assembly that catalyzes substrates in water, we show that the nanocage encapsulates a water cluster that is structurally and dynamically distinct from any known phase of water. It plays an important role in the driving force for guest encapsulation: The release of the highly unusual encapsulated water droplet creates a strong thermodynamic drive for the high-affinity binding of guests in aqueous solution for the supramolecular construct., Water under nanoconfinement at ambient conditions has exhibited low-dimensional ice formation and liquid–solid phase transitions, but with structural and dynamical signatures that map onto known regions of water’s phase diagram. Using terahertz (THz) absorption spectroscopy and ab initio molecular dynamics, we have investigated the ambient water confined in a supramolecular tetrahedral assembly, and determined that a dynamically distinct network of 9 ± 1 water molecules is present within the nanocavity of the host. The low-frequency absorption spectrum and theoretical analysis of the water in the Ga4L612− host demonstrate that the structure and dynamics of the encapsulated droplet is distinct from any known phase of water. A further inference is that the release of the highly unusual encapsulated water droplet creates a strong thermodynamic driver for the high-affinity binding of guests in aqueous solution for the Ga4L612− supramolecular construct.

Tracking the excitation of water molecules in the homogeneous liquid is challenging due to the ultrafast dissipation of rotational excitation energy through the hydrogen-bonded network. Here we demonstrate strong transient anisotropy of liquid water through librational excitation using single-color pump-probe experiments at 12.3 THz. We deduce a third order response of chi^3 exceeding previously reported values in the optical range by three orders of magnitude. Using a theory that replaces the nonlinear response with a material response property amenable to molecular dynamics simulation, we show that the rotationally damped motion of water molecules in the librational band is resonantly driven at this frequency, which could explain the enhancement of the anisotropy in the liquid by the external Terahertz field. By addition of salt (MgSO4), the hydration water is instead dominated by the local electric field of the ions, resulting in reduction of water molecules that can be dynamically perturbed by THz pulses.

A multi-technique approach, combining circular dichroism spectroscopy, ultraviolet resonance Raman spectroscopy and small angle scattering techniques, has been deployed to elucidate how the structural features of the human telomeric G-quadruplex d[A(GGGTTA)3GGG] (Tel22) change upon thermal unfolding. The system is studied both in the free form and when it is bound to Actinomycin D (ActD), an anticancer ligand with remarkable conformational flexibility. We find that at room temperature binding of Tel22 with ActD involves end-stacking upon the terminal G-tetrad. Structural evidence for drug-driven dimerization of a significant fraction of the G-quadruplexes is provided. When the temperature is raised, both free and bound Tel22 undergo melting through a multi-state process. We show that in the intermediate states of Tel22 the conformational equilibrium is shifted toward the (3+1) hybrid-type, while a parallel structure is promoted in the complex. The unfolded state of the free Tel22 is consistent with a self-avoiding random-coil conformation, whereas the high-temperature state of the complex is observed to assume a quite compact form. Such an unprecedented high-temperature arrangement is caused by the persistent interaction between Tel22 and ActD, which stabilizes compact conformations even in the presence of large thermal structural fluctuations.

Moringa oleifera is a fast-growing hygrophilic tree native to a humid sub-tropical region of India, now widely planted in many regions of the Southern Hemisphere characterized by low soil water availability. The widespread cultivation of this plant worldwide may have led to populations with different physiological and biochemical traits. In this work, the impact of water stress on the physiology and biochemistry of two M. oleifera populations, one from Chaco Paraguayo (PY) and one from Indian Andhra Pradesh (IA) region, was studied in a screenhouse experiment where the water stress treatment was followed by re-watering. Through transcriptome sequencing, 2201 potential genic simple sequence repeats were identified and used to confirm the genetic differentiation of the two populations. Both populations of M. oleifera reduced photosynthesis, water potential, relative water content and growth under drought, compared to control well-watered plants. A complete recovery of photosynthesis after re-watering was observed in both populations, but growth parameters recovered better in PY than in IA plants. During water stress, PY plants accumulated more secondary metabolites, especially β-carotene and phenylpropanoids, than IA plants, but IA plants invested more into xanthophylls and showed a higher de-epoxidation state of xanthophylls cycle that contributed to protect the photosynthetic apparatus. M. oleifera demonstrated a high genetic variability and phenotypic plasticity, which are key factors for adaptation to dry environments. A higher plasticity (e.g. in PY plants adapted to wet environments) will be a useful trait to endure recurrent but brief water stress episodes, whereas long-term investment of resources into secondary metabolism (e.g. in IA plants adapted to drier environments) will be a successful strategy to cope with prolonged periods of drought. This makes M. oleifera an important resource for agro-forestry in a climate change scenario.

Molecular mechanisms that are the base of the strategies adopted by Mediterranean plants to cope with the challenges imposed by limited or excessive solar radiation during the summer season have received limited attention. In our study, conducted on C. incanus plants growing in the shade or in full sunlight, we performed measurements of relevant physiological traits, such as leaf water potential, gas exchange and PSII photochemistry, RNA-Seq with de-novo assembly, and the analysis of differentially expressed genes. We also identified and quantified photosynthetic pigments, abscisic acid, and flavonoids. Here, we show major mechanisms regulating light perception and signaling which, in turn, sustain the shade avoidance syndrome displayed by the ‘sun loving’ C. incanus. We offer clear evidence of the detrimental effects of excessive light on both the assembly and the stability of PSII, and the activation of a suite of both repair and effective antioxidant mechanisms in sun-adapted leaves. For instance, our study supports the view of major antioxidant functions of zeaxanthin in sunny plants concomitantly challenged by severe drought stress. Finally, our study confirms the multiple functions served by flavonoids, both flavonols and flavanols, in the adaptive mechanisms of plants to the environmental pressures associated to Mediterranean climate.

Viele Naturphänomene hängen mit der Nukleation von CaCO3 zusammen, wobei die Rolle des Wassers bis jetzt weitgehend unklar war. Änderungen in der Absorption im THz-Frequenzbereich während der frühen Stadien der CaCO3-Nukleation belegen Veränderungen in den gekoppelten Bewegungen der hydratisierten Calcium- und Carbonat-Ionen. Der direkte Zusammenhang zwischen diesen Effekten und der kontinuierlichen Entwicklung des Ionenaktivitätsproduktes zeigt die Lage einer flüssig-flüssig-binodalen Grenze. Die Daten lassen stark darauf schließen, dass sich proto-strukturiertes, amorphes CaCO3 über die Verfestigung von anfänglich flüssigen Vorstufen bildet. Außerdem erhöhen Polycarboxylate, die flüssige Vorstufen von CaCO3 stabilisieren, die kinetische Stabilität des metastabilen flüssig-flüssigen Zustands signifikant, aber sie beeinflussen nicht die Lage der binodalen Grenze. Die mechanistische Bedeutung der Dynamik von Wassernetzwerken bei der Phasentrennung kann als alternativer Weg der Nukleation über Pränukleationscluster rationalisiert werden und ist wahrscheinlich allgemein für wässrige Systeme gültig. published

Many phenomena depend on CaCO3 nucleation where the role of water remains enigmatic. Changes in THz absorption during the early stages of CaCO3 nucleation evidence altered coupled motions of hydrated calcium and carbonate ions. The direct link between these changes and the continuous development of the ion activity product reveals the locus of a liquid–liquid binodal limit. The data strongly suggest that proto-structured amorphous CaCO3 forms through solidification of initially liquid precursors. Furthermore, polycarboxylates, which stabilize liquid precursors of CaCO3, significantly enhance the kinetic stability of the metastable liquid–liquid state, but they do not affect the locus of the binodal limit. The importance of water network dynamics in phase separation mechanisms can be understood based on the notions of the pre-nucleation cluster pathway, and is likely to be more general for aqueous systems. published

Whether flavonoids play significant antioxidant roles in plants challenged by photooxidative stress of different origin has been largely debated over the last few decades. A critical review of the pertinent literature and our experimentation as well, based on a free-of-scale approach, support an important antioxidant function served by flavonoids in plants exposed to a wide range of environmental stressors, the significance of which increases with the severity of stress. On the other side, some questions need conclusive answers when the putative antioxidant functions of plant flavonoids are examined at the level of both the whole-cell and cellular organelles. This partly depends upon a conclusive, robust, and unbiased definition of “a plant antioxidant”, which is still missing, and the need of considering the subcellular re-organization that occurs in plant cells in response to severe stress conditions. This likely makes our deterministic-based approach unsuitable to unveil the relevance of flavonoids as antioxidants in extremely complex biological systems, such as a plant cell exposed to an ever-changing stressful environment. This still poses open questions about how to measure the occurred antioxidant action of flavonoids. Our reasoning also evidences the need of contemporarily evaluating the changes in key primary and secondary components of the antioxidant defense network imposed by stress events of increasing severity to properly estimate the relevance of the antioxidant functions of flavonoids in an in planta situation. In turn, this calls for an in-depth analysis of the sub-cellular distribution of primary and secondary antioxidants to solve this still intricate matter.

Local and propagating vibrations occurring in DNA in the terahertz domain are essential for processes at the basis of cellular metabolism. Here we report the results of a study on the B-DNA terahertz dynamics where we combined high-resolution inelastic x-ray experiments and incoherent inelastic neutron scattering. By using two different high-hydration conditions we could study the effect of packing interactions between double helices, while by selecting Na and Cs counterions we could inspect how the mass loading affects the DNA low-frequency modes. The pattern of coherent excitation energies is well represented in terms of two branches, an acoustic-like one, with an associated propagation velocity of 3000 ± 100 m/s, and the other almost dispersionless at ~2 meV. This picture is also supported by the vibrational density of states projected on the hydrogen atoms. The acoustic-like mode is assigned to DNA excitations, despite its intriguing similarity with the analogous acoustic mode of bulk water at low wave-vector transfers. We also provide evidence for the intrahelical optic-like character of the low-energy mode, that we ascribe to large scale relative motions of DNA sub-domains.

Heterobasidion irregulare is a causal agent of root and butt-rot disease in conifers, and is native to North America. In 1944 it was introduced in central Italy in a Pinus pinea stand, where it shares the same niche with the native species Heterobasidion annosum. The introduction of a non-native pathogen may have significant negative effects on a naive host tree and the ecosystem in which it resides, requiring a better understanding of the system. We compared the spatio-temporal phenotypic, transcriptional and metabolic host responses to inoculation with the two Heterobasidion species in a large experiment with P. pinea seedlings. Differences in length of lesions at the inoculation site (IS), expression of host genes involved in lignin pathway and in cell rescue and defence, and analysis of terpenes at both IS and 12 cm above the IS (distal site, DS), were assessed at 3, 14 and 35 days post inoculation (dpi). Results clearly showed that both species elicit similar physiological and biochemical responses in P. pinea seedlings. The analysis of host transcripts and total terpenes showed differences between inoculation sites and between pathogen and mock inoculated plants. Both pathogen and mock inoculations induced antimicrobial peptide and phenylalanine ammonia-lyase overexpression at IS beginning at 3 dpi; while at DS all the analysed genes, except for peroxidase, were overexpressed at 14 dpi. A significantly higher accumulation of terpenoids was observed at 14 dpi at IS, and at 35 dpi at DS. The terpene blend at IS showed significant variation among treatments and sampling times, while no significant differences were ever observed in DS tissues. Based on our results, H. irregulare does not seem to have competitive advantages over the native species H. annosum in terms of pathogenicity towards P. pinea trees; this may explain why the non-native species has not widely spread over the 73 years since its putative year of introduction into central Italy.

Molecular mechanisms that are the base of the strategies adopted by Mediterranean plants to cope with the challenges imposed by limited or excessive solar radiation during the summer season have received limited attention. In our study, conducted on C. incanus plants growing in the shade or in full sunlight, we performed measurements of relevant physiological traits, such as leaf water potential, gas exchange and PSII photochemistry, RNA-Seq with de-novo assembly, and the analysis of differentially expressed genes. We also identified and quantified photosynthetic pigments, abscisic acid, and flavonoids. Here, we show major mechanisms regulating light perception and signaling which, in turn, sustain the shade avoidance syndrome displayed by the &lsquo, sun loving&rsquo, C. incanus. We offer clear evidence of the detrimental effects of excessive light on both the assembly and the stability of PSII, and the activation of a suite of both repair and effective antioxidant mechanisms in sun-adapted leaves. For instance, our study supports the view of major antioxidant functions of zeaxanthin in sunny plants concomitantly challenged by severe drought stress. Finally, our study confirms the multiple functions served by flavonoids, both flavonols and flavanols, in the adaptive mechanisms of plants to the environmental pressures associated to Mediterranean climate.

The distribution of plant species around the Mediterranean basin is a product of the influence of both geographical barriers and of climatic changes experienced during the Tertiary, with the transition from a warm to cool periods. Several species, once largely distributed across the Northern Hemisphere, retracted to refugial areas in southern Europe where they are described as Tertiary relicts. Platanus orientalis is a typical representative of Tertiary flora in southwest Eurasia; its distribution spreads from the Caucasus to the Mediterranean, with its western border in Southern Italy and Sicily. We analysed genetic diversity and differentiation in the centre and western part of its distribution range using nuclear microsatellites and compared genetic parameters between core and western populations. We found an overall decrease in genetic diversity estimates (heterozygosity, private allelic richness) from central towards western populations, with those from Southern Italy and Sicily showing the lowest values. The low level of genetic diversity probably results from historic range fragmentation experienced by P. orientalis in its westernmost distribution as confirmed by high level genetic isolation of these populations. Ornamental hybrids were genetically distinguished from P. orientalis as contained private alleles, indicating that gene flow with natural populations is rare. Population assignment and neighbour-joining (NJ) analysis of populations identified four groups belonging to two main phyletic groups (the Southern Italian-Sicilian-Balkan and Cretan-Bulgarian-Turkish lineages) that seem to have different biogeographic origin and also excluded an artificial origin for southern Italian and Sicilian populations as previously suggested. These results show that quantifying the genetic variation of a Tertiary relict in a geographical context, and the potential effect of hybridization with introduced ornamental hybrids, can provide useful insights on factors influencing population genetic structure. Such information is crucial to predict how such taxa will respond to increasing anthropogenic influence on the environment., Quantifying the genetic diversity of species is important for understanding their evolutionary history. We investigated the genetic diversity of a Tertiary relict, the tree Platanus orientalis, which has an unusual distribution in the Mediterranean with its largest populations in the east, and it is increasingly rare in the west. We found an overall decrease in genetic diversity from eastern to western populations, with populations in southern Italy containing the lowest levels of genetic diversity. As P. orientalis requires habitats with high moisture levels, these results provide crucial genetic information which may influence its potential to respond to environmental change.

Moringa oleifera is a fast-growing hygrophilic tree native to a humid sub-tropical region of India, now widely planted in many regions of the Southern Hemisphere characterized by low soil water availability. The widespread cultivation of this plant worldwide may have led to populations with different physiological and biochemical traits. In this work, the impact of water stress on the physiology and biochemistry of two M. oleifera populations, one from Chaco Paraguayo (PY) and one from Indian Andhra Pradesh (IA) region, was studied in a screenhouse experiment where the water stress treatment was followed by re-watering. Through transcriptome sequencing, 2201 potential genic simple sequence repeats were identified and used to confirm the genetic differentiation of the two populations. Both populations of M. oleifera reduced photosynthesis, water potential, relative water content and growth under drought, compared to control well-watered plants. A complete recovery of photosynthesis after re-watering was observed in both populations, but growth parameters recovered better in PY than in IA plants. During water stress, PY plants accumulated more secondary metabolites, especially β-carotene and phenylpropanoids, than IA plants, but IA plants invested more into xanthophylls and showed a higher de-epoxidation state of xanthophylls cycle that contributed to protect the photosynthetic apparatus. M. oleifera demonstrated a high genetic variability and phenotypic plasticity, which are key factors for adaptation to dry environments. A higher plasticity (e.g. in PY plants adapted to wet environments) will be a useful trait to endure recurrent but brief water stress episodes, whereas long-term investment of resources into secondary metabolism (e.g. in IA plants adapted to drier environments) will be a successful strategy to cope with prolonged periods of drought. This makes M. oleifera an important resource for agro-forestry in a climate change scenario.

Plants face excess light stress on daily as well as on seasonal basis. The excess of excitation energy on cellular organelles prone to reactive oxygen species (ROS) generation is further enhanced when plants growing in full sun concurrently experience drought and heat stress. These are the very conditions that promote the biosynthesis of a wide range of secondary metabolites. Plants display a highly integrated arsenal of ROS-detoxifying agents to keep ROS concentration under control for efficient signalling, while avoiding cell death. There is evidence that primary antioxidants, i.e., antioxidant enzymes and low molecular-weight antioxidants, such as ascorbic acid and glutathione, are depleted under a severe excess of radiant energy. Here we discuss about how relevant secondary metabolites, namely isoprene, carotenoids, and flavonoids may complement the function of primary antioxidants to avoid irreversible oxidative damage, when plants experience intense, even transient stress events. We offer evidence of how plants orchestrate daily the antioxidant machinery, when challenged against multiple environmental stresses. It is indeed conceivable that daily variations in sunlight irradiance and air temperature may greatly alter the effectiveness of primary and secondary ROS-detoxifying agents. Finally, we discuss about the possible inter-relation between isoprenoid and flavonoid metabolism in plants facing high light coupled with drought and heat stress, as a consequence of severe stress-induced redox imbalance.

Skin affections after sulfur mustard (SM) exposure include erythema, blister formation and severe inflammation. An antidote or specific therapy does not exist. Anti-inflammatory compounds as well as substances counteracting SM-induced cell death are under investigation. In this study, we investigated the benzylisoquinoline alkaloide berberine (BER), a metabolite in plants like berberis vulgaris, which is used as herbal pharmaceutical in Asian countries, against SM toxicity using a well-established in vitro approach. Keratinocyte (HaCaT) mono-cultures (MoC) or HaCaT/THP-1 co-cultures (CoC) were challenged with 100, 200 or 300 mM SM for 1 h. Post-exposure, both MoC and CoC were treated with 10, 30 or 50 mu M BER for 24 h. At that time, supernatants were collected and analyzed both for interleukine (IL) 6 and 8 levels and for content of adenylate-kinase (AK) as surrogate marker for cell necrosis. Cells were lysed and nucleosome formation as marker for late apoptosis was assessed. In parallel, AK in cells was determined for normalization purposes. BER treatment did not influence necrosis, but significantly decreased apoptosis. Anti-inflammatory effects were moderate, but also significant, primarily in CoC. Overall, BER has protective effects against SM toxicity in vitro. Whether this holds true should be evaluated in future in vivo studies.

Spectroscopy in the terahertz frequency regime is a sensitive tool to probe solvation-induced effects in aqueous solutions. Yet, a systematic understanding of spectral lineshapes as a result of distinct solvation contributions remains terra incognita. We demonstrate that modularization of amino acids in terms of functional groups allows us to compute their distinct contributions to the total terahertz response. Introducing the molecular cross-correlation analysis method provides unique access to these site-specific contributions. Equivalent groups in different amino acids lead to look-alike spectral contributions, whereas side chains cause characteristic but additive complexities. Specifically, hydrophilic solvation of the zwitterionic groups in valine and glycine leads to similar terahertz responses which are fully decoupled from the side chain. The terahertz response due to H-bonding within the large hydrophobic solvation shell of valine turns out to be nearly indistinguishable from that in bulk water in direct comparison to the changes imposed by the charged functional groups that form strong H-bonds with their hydration shells. Thus, the hydrophilic groups and their solvation shells dominate the terahertz absorption difference, while on the same intensity scale, the influence of hydrophobic water can be neglected.

Photosynthetic performance and the expression of genes involved in light signaling and the biosynthesis of isoprenoids and phenylpropanoids were analysed in green ('Tigullio', TIG) and red ('Red Rubin', RR) basil. The aim was to detect the physiological and molecular response mechanisms to high sunlight. The attenuation of blue-green light by epidermal anthocyanins was shown to evoke shade-avoidance responses with consequential effects on leaf morpho-anatomical traits and gas exchange performance. Red basil had a lower mesophyll conductance, partially compensated by the less effective control of stomatal movements, in comparison with TIG. Photosynthesis decreased more in TIG than in RR in high sunlight, because of larger stomatal limitations and the transient impairment of PSII photochemistry. The methylerythritol 4-phosphate pathway promoted above all the synthesis and de-epoxidation of violaxanthin-cycle pigments in TIG and of neoxanthin and lutein in RR. This enabled the green leaves to process the excess radiant energy effectively, and the red leaves to optimize light harvesting and photoprotection. The greater stomatal closure observed in TIG than in RR was due to enhanced abscisic acid (ABA) glucose ester deglucosylation and reduced ABA oxidation, rather than to superior de novo ABA synthesis. This study shows a strong competition between anthocyanin and flavonol biosynthesis, which occurs at the level of genes regulating the oxidation of the C2-C3 bond in the dihydro-flavonoid skeleton.