Identification and expression profile of the SMAX/SMXL family genes in chickpea and lentil provide important players of biotechnological interest involved in plant branching.

MAIN CONCLUSION: SMAX/SMXL family genes were successfully identified and characterized in the chickpea and lentil and gene expression data revealed several genes associated with the modulation of plant branching and powerful targets for use in transgenesis and genome editing. Strigolactones (SL) play essential roles in plant growth, rooting, development, and branching, and are associated with plant resilience to abiotic and biotic stress conditions. Likewise, karrikins (KAR) are "plant smoke-derived molecules" that act in a hormonal signaling pathway similar to SL playing an important role in seed germination and hairy root elongation. The SMAX/SMXL family genes are part of these two signaling pathways, in addition to some of these members acting in a still little known SL- and KAR-independent signaling pathway. To date, the identification and functional characterization of the SMAX/SMXL family genes has not been performed in the chickpea and lentil. In this study, nine SMAX/SMXL genes were systematically identified and characterized in the chickpea and lentil, and their expression profiles were explored under different unstressless or different stress conditions. After a comprehensive in silico characterization of the genes, promoters, proteins, and protein-protein interaction network, the expression profile for each gene was determined using a meta-analysis from the RNAseq datasets and complemented with real-time PCR analysis. The expression profiles of the SMAX/SMXL family genes were very dynamic in different chickpea and lentil organs, with some genes assuming a tissue-specific expression pattern. In addition, these genes were significantly modulated by different stress conditions, indicating that SMAX/SMXL genes, although working in three distinct signaling pathways, can act to modulate plant resilience. Most CaSMAX/SMXL and partner genes such as CaTiE1 and CaLAP1, have a positive correlation with the plant branching level, while most LcSMAX/SMXL genes were less correlated with the plant branching level. The SMXL6, SMXL7, SMXL8, TiE1, LAP1, BES1, and BRC1 genes were highlighted as powerful targets for use in transgenesis and genome editing aiming to develop chickpea and lentil cultivars with improved architecture. Therefore, this study presented a detailed characterization of the SMAX/SMXL genes in the chickpea and lentil, and provided new insights for further studies focused on each SMAX/SMXL gene.

Liquid Structure Scenario of the Archetypal Supramolecular Deep Eutectic Solvent: Heptakis(2,6-di-O-methyl)-ß-cyclodextrin/levulinic Acid.

The concept of supramolecular solvents has been recently introduced, and the extended liquid-state window accessible for mixtures of functionalized cyclodextrins (CDs) with hydrogen bond (HB) donor species, e.g., levulinic acid, led to the debut of supramolecular deep eutectic solvents (SUPRA-DES). These solvents retain CD's inclusion ability and complement it with enhanced solvation effectiveness due to an extended HB network. However, so far, these promising features were not rationalized in terms of a microscopic description, thus hindering a more complete capitalization. This is the first joint experimental and computational study on the archetypal SUPRA-DES: heptakis(2,6-di-O-methyl)-ß-CD/levulinic acid (1:27). We used X-ray scattering to probe CD's aggregation level and molecular dynamics simulation to determine the nature of interactions between SUPRA-DES components. We discover that CDs are homogeneously distributed in bulk and that HB interactions, together with the electrostatic ones, play a major role in determining mutual interaction between components. However, dispersive forces act in synergy with HB to accomplish a fundamental task in hindering hydrophobic interactions between neighbor CDs and maintaining the system homogeneity. The mechanism of mutual solvation of CD and levulinic acid is fully described, providing fundamental indications on how to extend the spectrum of SUPRA-DES combinations. Overall, this study provides the key to interpreting structural organization and solvation tunability in SUPRA-DES to extend the range of sustainable applications for these new, unique solvents.

Structural and Dynamic Disturbances Revealed by Molecular Dynamics Simulations Predict the Impact on Function of CCT5 Chaperonin Mutations Associated with Rare Severe Distal Neuropathies.

Mutations in genes encoding molecular chaperones, for instance the genes encoding the subunits of the chaperonin CCT (chaperonin containing TCP-1, also known as TRiC), are associated with rare neurodegenerative disorders. Using a classical molecular dynamics approach, we investigated the occurrence of conformational changes and differences in physicochemical properties of the CCT5 mutations His147Arg and Leu224Val associated with a sensory and a motor distal neuropathy, respectively. The apical domain of both variants was substantially but differently affected by the mutations, although these were in other domains. The distribution of hydrogen bonds and electrostatic potentials on the surface of the mutant subunits differed from the wild-type molecule. Structural and dynamic analyses, together with our previous experimental data, suggest that genetic mutations may cause different changes in the protein-binding capacity of CCT5 variants, presumably within both hetero- and/or homo-oligomeric complexes. Further investigations are necessary to elucidate the molecular pathogenic pathways of the two variants that produce the two distinct phenotypes. The data and clinical observations by us and others indicate that CCT chaperonopathies are more frequent than currently believed and should be investigated in patients with neuropathies.

Induction of 2-hydroxycatecholestrogens O-methylation: A missing puzzle piece in diagnostics and treatment of lung cancer.

Lung cancer is one of the most common cancers worldwide, causing nearly one million deaths each year. Herein, we present the effect of 2-methoxyestradiol (2-ME), the endogenous metabolite of 17ß-estradiol (E2), on non-small cell lung cancer (NSCLC) cells. We observed that 2-ME reduced the viability of lung adenocarcinoma in two-dimensional (2D) and three-dimensional (3D) spheroidal A549 cell culture models. Molecular modeling was carried out aiming to visualize amino acid residues within binding pockets of the acyl-protein thioesterases, namely 1 (APT1) and 2 (APT2), and thus to identify which ones were more likely involved in the interaction with 2-ME. Our findings suggest that 2-ME acts as an APT1 inhibitor enhancing protein palmitoylation and oxidative stress phenomena in the lung cancer cell. In order to support our data, metabolomics of blood serum from NSCLC patients was also performed. Moreover, computational analysis suggests that 2-ME as compared to other estrogen metabolism intermediates is relatively safe in terms of its possible non-receptor bioactivity within healthy human cells due to a very low electrophilic potential and hence no substantial risk of spontaneous covalent modification of biologically protective nucleophiles. We propose that 2-ME can be used as a selective tumor biomarker in the course of certain types of lung cancers and possibly as a therapeutic adjuvant or neoadjuvant.

Muscle Histopathological Abnormalities in a Patient With a CCT5 Mutation Predicted to Affect the Apical Domain of the Chaperonin Subunit.

Recognition of diseases associated with mutations of the chaperone system genes, e.g., chaperonopathies, is on the rise. Hereditary and clinical aspects are established, but the impact of the mutation on the chaperone molecule and the mechanisms underpinning the tissue abnormalities are not. Here, histological features of skeletal muscle from a patient with a severe, early onset, distal motor neuropathy, carrying a mutation on the CCT5 subunit (MUT) were examined in comparison with normal muscle (CTR). The MUT muscle was considerably modified; atrophy of fibers and disruption of the tissue architecture were prominent, with many fibers in apoptosis. CCT5 was diversely present in the sarcolemma, cytoplasm, and nuclei in MUT and in CTR and was also in the extracellular space; it colocalized with CCT1. In MUT, the signal of myosin appeared slightly increased, and actin slightly decreased as compared with CTR. Desmin was considerably delocalized in MUT, appearing with abnormal patterns and in precipitates. Alpha-B-crystallin and Hsp90 occurred at lower signals in MUT than in CTR muscle, appearing also in precipitates with desmin. The abnormal features in MUT may be the consequence of inactivity, malnutrition, denervation, and failure of protein homeostasis. The latter could be at least in part caused by malfunction of the CCT complex with the mutant CCT5 subunit. This is suggested by the results of the in silico analyses of the mutant CCT5 molecule, which revealed various abnormalities when compared with the wild-type counterpart, mostly affecting the apical domain and potentially impairing chaperoning functions. Thus, analysis of mutated CCT5 in vitro and in vivo is anticipated to provide additional insights on subunit involvement in neuromuscular disorders.

Sicilian Byzantine Icons through the Use of Non-Invasive Imaging Techniques and Optical Spectroscopy: The Case of the Madonna dell'Elemosina.

The iconographic heritage is one of the treasures of Byzantine art that have enriched the south of Italy, and Sicily in particular, since the early 16th century. In this work, the investigations of a Sicilian Icon of Greek-Byzantine origin, the Madonna dell'Elemosina, is reported for the first time. The study was carried out using mainly non-invasive imaging techniques (photography in reflectance and grazing visible light, UV fluorescence, infrared reflectography, radiography, and computed tomography) and spectroscopic techniques (X-ray fluorescence and infrared spectroscopy). The identification of the constituent materials provides a decisive contribution to the correct historical and artistic placement of the Icon, a treasure of the Eastern European historical community in Sicily. Some hidden details have also been highlighted. Most importantly, the information obtained enables us to define its conservation state, the presence of foreign materials, and to direct its protection and restoration.

Liquid Structure of a Water-in-Salt Electrolyte with a Remarkably Asymmetric Anion.

Water-in-salt systems, i.e., super-concentrated aqueous electrolytes, such as lithium bis(trifluoromethanesulfonyl)imide (21 mol/kgwater), have been recently discovered to exhibit unexpectedly large electrochemical windows and high lithium transference numbers, thus paving the way to safe and sustainable charge storage devices. The peculiar transport features in these electrolytes are influenced by their intrinsically nanoseparated morphology, stemming from the anion hydrophobic nature and manifesting as nanosegregation between anions and water domains. The underlying mechanism behind this structure-dynamics correlation is, however, still a matter of strong debate. Here, we enhance the apolar nature of the anions, exploring the properties of the aqueous electrolytes of lithium salts with a strongly asymmetric anion, namely, (trifluoromethylsulfonyl)(nonafluorobutylsulfonyl) imide. Using a synergy of experimental and computational tools, we detect a remarkable level of structural heterogeneity at a mesoscopic level between anion-rich and water-rich domains. Such a ubiquitous sponge-like, bicontinuous morphology develops across the whole concentration range, evolving from large fluorinated globules at high dilution to a percolating fluorous matrix intercalated by water nanowires at super-concentrated regimes. Even at extremely concentrated conditions, a large population of fully hydrated lithium ions, with no anion coordination, is detected. One can then derive that the concomitant coexistence of (i) a mesoscopically segregated structure and (ii) fully hydrated lithium clusters disentangled from anion coordination enables the peculiar lithium diffusion features that characterize water-in-salt systems.

Liquid structure and dynamics in the choline acetate:urea 1:2 deep eutectic solvent.

We report on the thermodynamic, structural, and dynamic properties of a recently proposed deep eutectic solvent, formed by choline acetate (ChAc) and urea (U) at the stoichiometric ratio 1:2, hereinafter indicated as ChAc:U. Although the crystalline phase melts at 36-38 °C depending on the heating rate, ChAc:U can be easily supercooled at sub-ambient conditions, thus maintaining at the liquid state, with a glass-liquid transition at about -50 °C. Synchrotron high energy x-ray scattering experiments provide the experimental data for supporting a reverse Monte Carlo analysis to extract structural information at the atomistic level. This exploration of the liquid structure of ChAc:U reveals the major role played by hydrogen bonding in determining interspecies correlations: both acetate and urea are strong hydrogen bond acceptor sites, while both choline hydroxyl and urea act as HB donors. All ChAc:U moieties are involved in mutual interactions, with acetate and urea strongly interacting through hydrogen bonding, while choline being mostly involved in van der Waals mediated interactions. Such a structural situation is mirrored by the dynamic evidences obtained by means of 1H nuclear magnetic resonance techniques, which show how urea and acetate species experience higher translational activation energy than choline, fingerprinting their stronger commitments into the extended hydrogen bonding network established in ChAc:U.

Pyrazole[3,4-d]pyrimidine derivatives loaded into halloysite as potential CDK inhibitors.

Uncontrolled cell proliferation is a hallmark of cancer as a result of rapid and deregulated progression through the cell cycle. The inhibition of cyclin-dependent kinases (CDKs) activities is a promising therapeutic strategy to block cell cycle of tumor cells. In this work we reported a new example of nanocomposites based on halloysite nanotubes (HNTs)/pyrazolo[3,4-d]pyrimidine derivatives (Si306 and Si113) as anticancer agents and CDK inhibitors. HNTs/Si306 and HNTs/Si113 nanocomposites were synthesized and characterized. The release kinetics were also investigated. Antitumoral activity was evaluated on three cancer cell lines (HeLa, MDA-MB-231 and HCT116) and the effects on cell cycle arrest in HCT116 cells were evaluated. Finally, molecular dynamics simulations were performed of the complexes between Si113 or Si306 and the active site of both CDK 1 and 2.

Quantum dots functionalised artificial peptides bioinspired to the D1 protein from the Photosystem II of Chlamydomonas reinhardtii for endocrine disruptor optosensing.

Herein we describe the design and synthesis of novel artificial peptides mimicking the plastoquinone binding niche of the D1 protein from the green photosynthetic alga Chlamydomonas reinhardtii, also able to bind herbicides. In particular, molecular dynamics (MD) simulations were performed to model in silico the behaviour of three peptides, D1Pep70-H, D1Pep70-S264K and D1Pep70-S268C, as genetic variants with different affinity towards the photosynthetic herbicide atrazine. Then the photosynthetic peptides were functionalised with quantum dots for the development of a hybrid optosensor for the detection of atrazine, one of the most employed herbicides for weed control in agriculture as well as considered as a putative endocrine disruptor case study. The excellent agreement between computational and experimental results self consistently shows resistance or super-sensitivity toward the atrazine target, with detection limits in the µg/L concentration range, meeting the requirements of E.U. legislation.

Green Tea Catechins Induce Inhibition of PTP1B Phosphatase in Breast Cancer Cells with Potent Anti-Cancer Properties: In Vitro Assay, Molecular Docking, and Dynamics Studies.

The catechins derived from green tea possess antioxidant activity and may have a potentially anticancer effect. PTP1B is tyrosine phosphatase that is oxidative stress regulated and is involved with prooncogenic pathways leading to the formation of a.o. breast cancer. Here, we present the effect of selected green tea catechins on enzymatic activity of PTP1B phosphatase and viability of MCF-7 breast cancer cells. We showed also the computational analysis of the most effective catechin binding with a PTP1B molecule. We observed that epigallocatechin, epigallocatechin gallate, epicatechin, and epicatechin gallate may decrease enzymatic activity of PTP1B phosphatase and viability of MCF-7 cells. Conclusions: From the tested compounds, epigallocatechin and epigallocatechin gallate were the most effective inhibitors of the MCF-7 cell viability. Moreover, epigallocatechin was also the strongest inhibitor of PTP1B activity. Computational analysis allows us also to conclude that epigallocatechin is able to interact and bind to PTP1B. Our results suggest also the most predicted binding site to epigallocatechin binding to PTP1B.

Identification of New Antimicrobial Peptides from Mediterranean Medical Plant Charybdis pancration (Steinh.) Speta.

The present work was designed to identify and characterize novel antimicrobial peptides (AMPs) from Charybdis pancration (Steinh.) Speta, previously named Urginea maritima, is a Mediterranean plant, well-known for its biological properties in traditional medicine. Polypeptide-enriched extracts from different parts of the plant (roots, leaves and bulb), never studied before, were tested against two relevant pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. With the aim of identifying novel natural AMPs, peptide fraction displaying antimicrobial activity (the bulb) that showed minimum inhibitory concentration (MICs) equal to 30 µg/mL against the above mentioned strains, was analysed by high-resolution mass spectrometry and database search. Seventeen peptides, related to seven proteins present in the investigated database, were described. Furthermore, we focused on three peptides, which due to their net positive charge, have a better chance to be AMPs and they were investigated by molecular modelling approaches, in order to shed light on the solution properties of their equilibrium structures. Some of new detected peptides could represent a good platform for the development of new antimicrobials in the fight against antibiotic resistance phenomenon.

A Novel CCT5 Missense Variant Associated with Early Onset Motor Neuropathy.

Diseases associated with acquired or genetic defects in members of the chaperoning system (CS) are increasingly found and have been collectively termed chaperonopathies. Illustrative instances of genetic chaperonopathies involve the genes for chaperonins of Groups I (e.g., Heat shock protein 60, Hsp60) and II (e.g., Chaperonin Containing T-Complex polypeptide 1, CCT). Examples of the former are hypomyelinating leukodystrophy 4 (HLD4 or MitCHAP60) and hereditary spastic paraplegia (SPG13). A distal sensory mutilating neuropathy has been linked to a mutation [p.(His147Arg)] in subunit 5 of the CCT5 gene. Here, we describe a new possibly pathogenic variant [p.(Leu224Val)] of the same subunit but with a different phenotype. This yet undescribed disease affects a girl with early onset demyelinating neuropathy and a severe motor disability. By whole exome sequencing (WES), we identified a homozygous CCT5 c.670C>G p.(Leu224Val) variant in the CCT5 gene. In silico 3D-structure analysis and bioinformatics indicated that this variant could undergo abnormal conformation and could be pathogenic. We compared the patient's clinical, neurophysiological and laboratory data with those from patients carrying p.(His147Arg) in the equatorial domain. Our patient presented signs and symptoms absent in the p.(His147Arg) cases. Molecular dynamics simulation and modelling showed that the Leu224Val mutation that occurs in the CCT5 intermediate domain near the apical domain induces a conformational change in the latter. Noteworthy is the striking difference between the phenotypes putatively linked to mutations in the same CCT subunit but located in different structural domains, offering a unique opportunity for elucidating their distinctive roles in health and disease.

Modification of DNA structure by reactive nitrogen species as a result of 2-methoxyestradiol-induced neuronal nitric oxide synthase uncoupling in metastatic osteosarcoma cells.

2-methoxyestradiol (2-ME) is a physiological anticancer compound, metabolite of 17ß-estradiol. Previously, our group evidenced that from mechanistic point of view one of anticancer mechanisms of action of 2-ME is specific induction and nuclear hijacking of neuronal nitric oxide synthase (nNOS), resulting in local generation of nitro-oxidative stress and finally, cancer cell death. The current study aims to establish the substantial mechanism of generation of reactive nitrogen species by 2-ME. We further achieved to identify the specific reactive nitrogen species involved in DNA-damaging mechanism of 2-ME. The study was performed using metastatic osteosarcoma 143B cells. We detected the release of biologically active (free) nitric oxide (•NO) with concurrent measurements of peroxynitrite (ONOO-) in real time in a single cell of 143B cell line by using •NO/ONOO- sensitive microsensors after stimulation with calcium ionophore. Detection of nitrogen dioxide (•NO2) and determination of chemical rate constants were carried out by a stopped-flow technique. The affinity of reactive nitrogen species toward the guanine base of DNA was evaluated by density functional theory calculations. Expression and localization of nuclear factor NF-kB was determined using imaging cytometry, while cell viability assay was evaluated by MTT assay. Herein, we presented that 2-ME triggers pro-apoptotic signalling cascade by increasing cellular reactive nitrogen species overproduction - a result of enzymatic uncoupling of increased nNOS protein levels. In particular, we proved that ONOO- and •NO2 directly formed from peroxynitrous acid (ONOOH) and/or by auto-oxidation of •NO, are inducers of DNA damage in anticancer mechanism of 2-ME. Specifically, the affinity of reactive nitrogen species toward the guanine base of DNA, evaluated by density functional theory calculations, decreased in the order: ONOOH > ONOO- > â€¢NO2 > â€¢NO. Therefore, we propose to consider the specific inducers of nNOS as an effective tool in the field of chemotherapy.

Structural Features of ß-Cyclodextrin Solvation in the Deep Eutectic Solvent, Reline.

The inherently amphiphilic nature of native cyclodextrins (CDs) determines their peculiar molecular encapsulation features, enabling applications such as targeted drug nanodelivery, aroma protection, etc. On the contrary, it may also lead to poor solubility in water and other organic solvents and to potentially detrimental flocking in these media, thus posing limitations to more extensive usage. Here we use small angle X-ray scattering to show that deep eutectic solvent reline (1:2 choline chloride:urea) succeeds in dissolving large amounts of ß-CD (at least 800 mg/mL, compared with the solubility in water of 18 mg/mL), without aggregation phenomena occurring. At the microscopic level, molecular dynamics simulations highlight the complex interplay of hydrogen bonding-mediated hydrophilic interactions and hydrophobic force mitigation occurring between ß-CD and reline components, leading to energetically favorable ß-CD solvation. The possibility of achieving very high concentration conditions for unaggregated ß-CD in an environmentally responsible media, such as reline, can open the way to new, so far unpredictable applications, addressing multiple societal challenges.

Structural features of selected protic ionic liquids based on a super-strong base.

Protic ionic liquids (PIL) were prepared from a super-strong base 1,7-diazabicyclo[5.4.0]undec-7-ene (DBU) and super-strong acids, trifluoromethane sulfonic acid (TfOH), and (trifluoromethanesulfonyl)-(nonafluorobutylsulfonyl)imide, (IM14H), ([DBUH][TfO] and [DBUH][IM14], respectively; the latter for the first time) and their chemical and physical properties and structural features have been explored using a synergy of experimental and computational tools. The short range order in neat DBU, as well as the long range structural correlations induced by charge correlation and hydrogen bonding interactions in the ionic liquids, have been explored under ambient conditions, where these compounds are proposed for a variety of applications. Similar to other [DBUH]-based PILs, the probed ones behave as good ionic liquids. Molecular dynamics-rationalised X-ray diffraction patterns show the major role played by hydrogen bonding in affecting morphology in these systems. Additionally, we find further evidence for the existence of fluorous domains in [DBUH][IM14], thus potentially extending the range of applications for these PILs.

Microscopic Structural and Dynamic Features in Triphilic Room Temperature Ionic Liquids.

Here we report a thorough investigation of the microscopic and mesoscopic structural organization in a series of triphilic fluorinated room temperature ionic liquids, namely [1-alkyl,3-methylimidazolium][(trifluoromethanesulfonyl)(nonafluorobutylsulfonyl)imide], with alkyl = ethyl, butyl, octyl ([Cnmim][IM14], n = 2, 4, 8), based on the synergic exploitation of X-ray and Neutron Scattering and Molecular Dynamics simulations. This study reveals the strong complementarity between X-ray/neutron scattering in detecting the complex segregated morphology in these systems at mesoscopic spatial scales. The use of MD simulations delivering a very good agreement with experimental data allows us to gain a robust understanding of the segregated morphology. The structural scenario is completed with determination of dynamic properties accessing the diffusive behavior and a relaxation map is provided for [C2mim][IM14] and [C8mim][IM14], highlighting their natures as fragile glass formers.

Communication: Anion-specific response of mesoscopic organization in ionic liquids upon pressurization.

One of the outstanding features of ionic liquids is their inherently hierarchical structural organization at mesoscopic spatial scales. Recently experimental and computational studies showed the fading of this feature when pressurising. Here we use simulations to show that this effect is not general: appropriate anion choice leads to an obstinate resistance against pressurization.

Mesoscopic organization in ionic liquids.

We discuss some published results and provide new observations concerning the high level of structural complexity that lies behind the nanoscale correlations in ionic liquids (ILs) and their mixtures with molecular liquids. It turns out that this organization is a consequence of the hierarchical construction on both spatial (from ångström to several nanometer) and temporal (from fraction of picosecond to hundreds of nanosecond) scales, which requires joint use of experimental and computational tools.

Emerging Evidences of Mesoscopic-Scale Complexity in Neat Ionic Liquids and Their Mixtures.

Ionic liquids (ILs) represent a blooming class of continuously developing advanced materials, with the aiming of a green chemical industry. Their appealing physical and chemical properties are largely influenced by their micro- and mesoscopic structure that is known to possess a high degree of hierarchical organization. High-impact application fields are largely affected by the complex morphology of neat ionic liquids and their mixtures. This Perspective highlights new arising research directions that point to an enhanced level of structural complexity in several IL-based systems, including mixtures. The latter represent a change in paradigm in the approach to formulate new, task-specific IL-based media, and the reported phenomenology has the potential to further expand their range of applications by calling for a revisitation of the nature of interactions in these exciting media.