Deciphering the Role of Trehalose in Chroococcidiopsis sp. 029's High-Desiccation Resistance: Sequence Determination, Structural Modelling and Simulative Analysis of the 30S Ribosomal Subunit.

Desert strains of the genus Chroococcidiopsis are among the most desiccation-resistant cyanobacteria capable of anhydrobiosis. The accumulation of two sugars, sucrose and trehalose, facilitates the entrance of anhydrobiotes into a reversible state of dormancy by stabilizing cellular components upon water removal. This study aimed to evaluate, at the atomistic level, the role of trehalose in desiccation resistance by using as a model system the 30S ribosomal subunit of the desert cyanobacterium Chroococcidiopsis sp. 029. Molecular dynamic simulations provided atomistic evidence regarding its protective role on the 30S molecular structure. Trehalose forms an enveloping shell around the ribosomal subunit and stabilizes the structures through a network of direct interactions. The simulation confirmed that trehalose actively interacts with the 30S ribosomal subunit and that, by replacing water molecules, it ensures ribosomal structural integrity during desiccation, thus enabling protein synthesis to be carried out upon rehydration.

Profile and potential bioactivity of the miRNome and metabolome expressed in Malva sylvestris L. leaf and flower.

Malva sylvestris L. (common mallow) is a plant species widely used in phytotherapy and ethnobotanical practices since time immemorial. Characterizing the components of this herb might promote a better comprehension of its biological effects on the human body but also favour the identification of the molecular processes that occur in the plant tissues. Thus, in the present contribution, the scientific knowledge about the metabolomic profile of the common mallow was expanded. In particular, the phytocomplex of leaves and flowers from this botanical species and the extraction capacity of different concentrations of ethanol (i.e., 95%, 70%, 50%, and 0%; v/v in ddH2O) for it were investigated by spectrophotometric and chromatographic approaches. In detail, 95% ethanol extracts showed the worst capacity in isolating total phenols and flavonoids, while all the hydroalcoholic samples revealed a specific ability in purifying the anthocyanins. HPLC-DAD system detected and quantified 20 phenolic secondary metabolites, whose concentration in the several extracts depended on their own chemical nature and the percentage of ethanol used in the preparation. In addition, the stability of the purified phytochemicals after resuspension in pure ddH2O was also proved, considering a potential employment of them in biological/medical studies which include in vitro and in vivo experiments on mammalian models. Here, for the first time, the expressed miRNome in M. sylvestris was also defined by Next Generation Sequencing, revealing the presence of 33 microRNAs (miRNAs), 10 typical for leaves and 2 for flowers. Then, both plant and human putative mRNA targets for the detected miRNAs were predicted by bioinformatics analyses, with the aim to clarify the possible role of these small nucleic acids in the common mallow plant tissues and to try to understand if they could exert a potential cross-kingdom regulatory activity on the human health. Surprisingly, our investigations revealed that 19 miRNAs out of 33 were putatively able to modulate, in the plant cells, the expression of various chromosome scaffold proteins. In parallel, we found, in the human transcriptome, a total of 383 mRNAs involved in 5 fundamental mammalian cellular processes (i.e., apoptosis, senescence, cell-cycle, oxidative stress, and invasiveness) that theoretically could be bound and regulated by M. sylvestris miRNAs. The evidence collected in this work would suggest that the beneficial properties of the use of M. sylvestris, documented by the folk medicine, are probably linked to their content of miRNAs and not only to the action of phytochemicals (e.g., anthocyanins). This would open new perspectives about the possibility to develop gene therapies based on miRNAs isolated from medicinal plants, including M. sylvestris.

Deciphering the Broad Antimicrobial Activity of Melaleuca alternifolia Tea Tree Oil by Combining Experimental and Computational Investigations.

Tea Tree Oil (TTO) is an essential oil obtained from the distillation of Melaleuca alternifolia leaves and branches. Due to its beneficial properties, TTO is widely used as an active ingredient in antimicrobial preparations for topical use or in cosmetic products and contains about 100 different compounds, with terpinen-4-ol, γ-terpinene and 1,8-cineole (or eucalyptol) being the molecules most responsible for its biological activities. In this work, the antimicrobial activity of whole TTO and these three major components was evaluated in vitro against fungi, bacteria and viruses. Molecular dynamics simulations were carried out on a bacterial membrane model and a Coxsackievirus B4 viral capsid, to propose an atomistic explanation of their mechanism of action. The obtained results indicate that the strong antimicrobial activity of TTO is attributable to the induction of an altered membrane functionality, mediated by the incorporation of its components within the lipid bilayer, and to a possible ability of the compounds to bind and alter the structural properties of the viral capsid.

Potential Use of Tea Tree Oil as a Disinfectant Agent against Coronaviruses: A Combined Experimental and Simulation Study.

The COVID-19 pandemic has highlighted the relevance of proper disinfection procedures and renewed interest in developing novel disinfectant materials as a preventive strategy to limit SARS-CoV-2 contamination. Given its widely known antibacterial, antifungal, and antiviral properties, Melaleuca alternifolia essential oil, also named Tea tree oil (TTO), is recognized as a potential effective and safe natural disinfectant agent. In particular, the proposed antiviral activity of TTO involves the inhibition of viral entry and fusion, interfering with the structural dynamics of the membrane and with the protein envelope components. In this study, for the first time, we demonstrated the virucidal effects of TTO against the feline coronavirus (FCoVII) and the human coronavirus OC43 (HCoV-OC43), both used as surrogate models for SARS-CoV-2. Then, to atomistically uncover the possible effects exerted by TTO compounds on the outer surface of the SARS-CoV-2 virion, we performed Gaussian accelerated Molecular Dynamics simulations of a SARS-CoV-2 envelope portion, including a complete model of the Spike glycoprotein in the absence or presence of the three main TTO compounds (terpinen-4-ol, γ-terpinene, and 1,8-cineole). The obtained results allowed us to hypothesize the mechanism of action of TTO and its possible use as an anti-coronavirus disinfectant agent.

Antifungal Effect of All-trans Retinoic Acid against Aspergillus fumigatus In Vitro and in a Pulmonary Aspergillosis In Vivo Model.

Aspergillus fumigatus is the most common opportunistic fungal pathogen and causes invasive pulmonary aspergillosis (IPA), with high mortality among immunosuppressed patients. The fungistatic activity of all-trans retinoic acid (ATRA) has been recently described in vitro We evaluated the efficacy of ATRA in vivo and its potential synergistic interaction with other antifungal drugs. A rat model of IPA and in vitro experiments were performed to assess the efficacy of ATRA against Aspergillus in association with classical antifungal drugs and in silico studies used to clarify its mechanism of action. ATRA (0.5 and 1 mM) displayed a strong fungistatic activity in Aspergillus cultures, while at lower concentrations, synergistically potentiated fungistatic efficacy of subinhibitory concentration of amphotericin B (AmB) and posaconazole (POS). ATRA also enhanced macrophagic phagocytosis of conidia. In a rat model of IPA, ATRA reduced mortality similarly to posaconazole. Fungistatic efficacy of ATRA alone and synergistically with other antifungal drugs was documented in vitro, likely by inhibiting fungal heat shock protein 90 (Hsp90) expression and Hsp90-related genes. ATRA treatment reduced mortality in a model of IPA in vivo Those findings suggest ATRA as a suitable fungistatic agent that can also reduce dosage and adverse reactions of classical antifungal drugs and add to the development of new therapeutic strategies against IPA and systemic fungal infections.

Natural Compounds as Therapeutic Agents: The Case of Human Topoisomerase IB.

Natural products are widely used as source for drugs development. An interesting example is represented by natural drugs developed against human topoisomerase IB, a ubiquitous enzyme involved in many cellular processes where several topological problems occur due the formation of supercoiled DNA. Human topoisomerase IB, involved in the solution of such problems relaxing the DNA cleaving and religating a single DNA strand, represents an important target in anticancer therapy. Several natural compounds inhibiting or poisoning this enzyme are under investigation as possible new drugs. This review summarizes the natural products that target human topoisomerase IB that may be used as the lead compounds to develop new anticancer drugs. Moreover, the natural compounds and their derivatives that are in clinical trial are also commented on.

In Vitro and In Silico Characterization of an Antimalarial Compound with Antitumor Activity Targeting Human DNA Topoisomerase IB.

Human DNA topoisomerase IB controls the topological state of supercoiled DNA through a complex catalytic cycle that consists of cleavage and religation reactions, allowing the progression of fundamental DNA metabolism. The catalytic steps of human DNA topoisomerase IB were analyzed in the presence of a drug, obtained by the open-access drug bank Medicines for Malaria Venture. The experiments indicate that the compound strongly and irreversibly inhibits the cleavage step of the enzyme reaction and reduces the cell viability of three different cancer cell lines. Molecular docking and molecular dynamics simulations suggest that the drug binds to the human DNA topoisomerase IB-DNA complex sitting inside the catalytic site of the enzyme, providing a molecular explanation for the cleavage-inhibition effect. For all these reasons, the aforementioned drug could be a possible lead compound for the development of an efficient anti-tumor molecule targeting human DNA topoisomerase IB.

Post Zygotic, Somatic, Deletion in KERATIN 1 V1 Domain Generates Structural Alteration of the K1/K10 Dimer, Producing a Monolateral Palmar Epidermolytic Nevus.

Palmoplantar keratodermas (PPKs) are characterized by thickness of stratum corneum and epidermal hyperkeratosis localized in palms and soles. PPKs can be epidermolytic (EPPK) or non epidermolytic (NEPPK). Specific mutations of keratin 16 (K16) and keratin 1 (K1) have been associated to EPPK, and NEPPK. Cases of mosaicism in PPKs due to somatic keratin mutations have also been described in scientific literature. We evaluated a patient presenting hyperkeratosis localized monolaterally in the right palmar area, characterized by linear yellowish hyperkeratotic lesions following the Blaschko lines. No other relatives of the patient showed any dermatological disease. Light and confocal histological analysis confirmed the presence of epidermolityic hyperkeratosis. Genetic analysis performed demonstrates the heterozygous deletion NM_006121.4:r.274_472del for a total of 198 nucleotides, in KRT1 cDNA obtained by a palmar lesional skin biopsy, corresponding to the protein mutation NP_006112.3:p.Gly71_Gly137del. DNA extracted from peripheral blood lymphocytes did not display the presence of the mutation. These results suggest a somatic mutation causing an alteration in K1 N-terminal variable domain (V1). The deleted sequence involves the ISIS subdomain, containing a lysine residue already described as fundamental for epidermal transglutaminases in the crosslinking of IF cytoskeleton. Moreover, a computational analysis of the wild-type and V1-mutated K1/K10 keratin dimers, suggests an unusual interaction between these keratin filaments. The mutation taster in silico analysis also returned a high probability for a deleterious mutation. These data demonstrate once again the importance of the head domain (V1) of K1 in the formation of a functional keratinocyte cytoskeleton. Moreover, this is a further demonstration of the presence of somatic mutations arising in later stages of the embryogenesis, generating a mosaic phenotype.

Engineering a responsive DNA triple helix into an octahedral DNA nanostructure for a reversible opening/closing switching mechanism: a computational and experimental integrated study.

We propose an experimental and simulative approach to study the effect of integrating a DNA functional device into a large-sized DNA nanostructure. We selected, as a test bed, a well-known and characterized pH-dependent clamp-switch, based on a parallel DNA triple helix, to be integrated into a truncated octahedral scaffold. We designed, simulated and experimentally characterized two different functionalized DNA nanostructures, with and without the presence of a spacer between the scaffold and the functional elements. The experimental and simulative data agree in validating the need of a spacer for the occurrence of the pH dependent switching mechanism. The system is fully reversible and the switching can be monitored several times without any perturbation, maintaining the same properties of the isolated clamp switch in solution.

Dynamical Behavior of the Human Ferroportin Homologue from Bdellovibrio bacteriovorus: Insight into the Ligand Recognition Mechanism.

Members of the major facilitator superfamily of transporters (MFS) play an essential role in many physiological processes such as development, neurotransmission, and signaling. Aberrant functions of MFS proteins are associated with several diseases, including cancer, schizophrenia, epilepsy, amyotrophic lateral sclerosis and Alzheimer's disease. MFS transporters are also involved in multidrug resistance in bacteria and fungi. The structures of most MFS members, especially those of members with significant physiological relevance, are yet to be solved. The lack of structural and functional information impedes our detailed understanding, and thus the pharmacological targeting, of these transporters. To improve our knowledge on the mechanistic principles governing the function of MSF members, molecular dynamics (MD) simulations were performed on the inward-facing and outward-facing crystal structures of the human ferroportin homologue from the Gram-negative bacterium Bdellovibrio bacteriovorus (BdFpn). Several simulations with an excess of iron ions were also performed to explore the relationship between the protein's dynamics and the ligand recognition mechanism. The results reinforce the existence of the alternating-access mechanism already described for other MFS members. In addition, the reorganization of salt bridges, some of which are conserved in several MFS members, appears to be a key molecular event facilitating the conformational change of the transporter.

Probing the Functional Topology of a pH-Dependent Triple Helix DNA Nanoswitch Family through Gaussian Accelerated MD Simulation.

The topology of a pH-dependent triple helix DNA nanoswitch family has been characterized through simulative analysis to evaluate the efficiency of the switching mechanism varying the length of the loop connecting the two strands forming the double helix portion. In detail, the system is formed by a double helix made by two six base complementary sequences, connected by one loop having an increasing number of thymidines, namely 5, 7, or 9. The triplex-forming sequence made by six bases, connected to the double helix through a constant 25 base loop, interacts at pH 5.0 through Hoogsteen hydrogen bonds with one strand of the double helical region. We demonstrate, through molecular dynamics simulation, that the thymidine loop length exerts a fine regulatory role for the stability of the triple helix structure and is critical in modulating the switching mechanism triggered by the pH increase.

In Silico and In Cell Analysis of Openable DNA Nanocages for miRNA Silencing.

A computational and experimental integrated approach was applied in order to study the effect of engineering four DNA hairpins into an octahedral truncated DNA nanocage, to obtain a nanostructure able to recognize and bind specific oligonucleotide sequences. Modeling and classical molecular dynamics simulations show that the new H4-DNA nanocage maintains a stable conformation with the closed hairpins and, when bound to complementary oligonucleotides produces an opened conformation that is even more stable due to the larger hydrogen bond number between the hairpins and the oligonucleotides. The internal volume of the open conformation is much larger than the closed one, switching from 370 to 650 nm3, and the predicted larger conformational change is experimentally detectable by gel electrophoresis. H4-DNA nanocages display high stability in serum, can efficiently enter the cells where they are stable and maintain the ability to bind, and sequester an intracellular-specific oligonucleotide. Moreover, H4-DNA nanocages, modified in order to recognize the oncogenic miR21, are able to seize miRNA molecules inside cells in a selective manner.

Luteolin-7-O-ß-d-Glucoside Inhibits Cellular Energy Production Interacting with HEK2 in Keratinocytes.

Flavonoids have been demonstrated to affect the activity of many mammalian enzyme systems. Their functional phenolic groups are able to mediate antioxidant effects by scavenging free radicals. Molecules of this class have been found able to modulate the activity of kinases, phospholipase A2, cyclooxygenases, lipoxygenase, glutathione S-transferase, and many others. Recently, it has been demonstrated that luteolin, in the form of Luteolin-7-O-ß-d-glucoside (LUT-7G) is able to induce the keratinocyte differentiation process in vitro. This flavonoid is able to counteract the proliferative effects of IL-22/IL6 pathway by the inhibition of STAT3 activity also in vivo in a psoriatic mouse model. Observations on energy metabolism changes of differentiating cells led us to perform a complete metabolomics analysis using human primary keratinocytes treated with LUT-7G. Our results show that LUT-7G, is not only able to impair the nuclear translocation of STAT3, but it also blocks the energy metabolism pathway, depressing the glycolytic and Krebs pathway by the inhibition of hexokinase 2 activity. These data confirm that LUT-7G can be proposed as a potential candidate for the treatment of inflammatory and proliferative diseases, but its role as a hexokinase 2 (HEK2) inhibitor opens new perspectives in nutritional science, and especially in cancer therapy, in which the inhibition of the Warburg effect could be relevant.

Succinic semialdehyde dehydrogenase deficiency: The combination of a novel ALDH5A1 gene mutation and a missense SNP strongly affects SSADH enzyme activity and stability.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare autosomal recessive metabolic disorder of GABA catabolism. SSADH is a mitochondrial homotetrameric enzyme encoded by ALDH5A1 gene. We report the molecular characterization of ALDH5A1 gene in an Italian SSADHD patient, showing heterozygosity for four missense mutations: c.526G>A (p.G176R), c.538C>T (p.H180Y), c.709G>T (p.A237S) and c.1267A>T (p.T423S), the latter never described so far. The patient inherited c.526A in cis with c.538T from the mother and c.709T in cis with c.1267T from the father. To explore the effects of the two allelic arrangements on SSADH activity and protein level, wild type, single or double mutated cDNA constructs were expressed in a cell system. The p.G176R change, alone or in combination with p.H180Y, causes the abolishment of enzyme activity. Western blot analysis showed a strongly reduced amount of the p.176R-p.180Y double mutant protein, suggesting increased degradation. Indeed, in silico analyses confirmed high instability of this mutant homotetramer. Enzyme activity relative to the other p.423S-p.237S double mutant is around 30% of wt. Further in silico analyses on all the possible combinations of mutant monomers suggest the lowest stability for the tetramer constituted by p.176R-p.180Y monomers and the highest stability for that constituted by p.237S-p.423S monomers. The present study shows that when a common SNP, associated with a slight reduction of SSADH activity, is inherited in cis with a mutation showing no consequences on the enzyme function, the activity is strongly affected. In conclusion, the peculiar arrangement of four missense mutations occurring in this patient is responsible for the SSADHD phenotype.

Selective targeting and degradation of doxorubicin-loaded folate-functionalized DNA nanocages.

Selective targeting is a crucial property of nanocarriers used for drug delivery in cancer therapy. We generated biotinylated octahedral DNA nanocages functionalized with folic acid through bio-orthogonal conjugation chemistry. Molecular modelling indicated that a distance of about 2.5 nm between folic acid and DNA nanocage avoids steric hindrance with the folate receptor. HeLa cells, a folate receptor positive tumour cell line, internalize folate-DNA nanocages with efficiency greater than 40 times compared to cells not expressing the folate receptors. Functionalized DNA nanocages are highly stable, not cytotoxic and can be efficiently loaded with the chemotherapeutic agent doxorubicin. After entry into cells, doxorubicin-loaded nanoparticles are confined in vesicular structures, indicating that DNA nanocages traffic through the endocytic pathway. Doxorubicin release from loaded DNA cages, facilitated by low pH of endocytic vesicles, induces toxic pathways that, besides selectively killing folate receptor-positive cancer cells, leads to cage degradation avoiding nanoparticles accumulation inside cells.

Multiple molecular dynamics simulations of human LOX-1 and Trp150Ala mutant reveal the structural determinants causing the full deactivation of the receptor.

Multiple classical molecular dynamics simulations have been applied to the human LOX-1 receptor to clarify the role of the Trp150Ala mutation in the loss of binding activity. Results indicate that the substitution of this crucial residue, located at the dimer interface, markedly disrupts the wild-type receptor dynamics. The mutation causes an irreversible rearrangement of the subunits interaction pattern that in the wild-type protein allows the maintaining of a specific symmetrical motion of the monomers. The subunits dislocation determines a loss of linearity of the arginines residues composing the basic spine and a consequent alteration of the long-range electrostatic attraction of the substrate. Moreover, the anomalous subunits arrangement observed in the mutated receptor also affects the integrity of the hydrophobic tunnel, actively involved in the short-range hydrophobic recognition of the substrate. The combined effect of these structural rearrangements generates the impairing of the receptor function.

Simulative and Experimental Characterization of a pH-Dependent Clamp-like DNA Triple-Helix Nanoswitch.

Here we couple experimental and simulative techniques to characterize the structural/dynamical behavior of a pH-triggered switching mechanism based on the formation of a parallel DNA triple helix. Fluorescent data demonstrate the ability of this structure to reversibly switch between two states upon pH changes. Two accelerated, half microsecond, MD simulations of the system having protonated or unprotonated cytosines, mimicking the pH 5.0 and 8.0 conditions, highlight the importance of the Hoogsteen interactions in stabilizing the system, finely depicting the time-dependent disruption of the hydrogen bond network. Urea-unfolding experiments and MM/GBSA calculations converge in indicating a stabilization energy at pH 5.0, 2-fold higher than that observed at pH 8.0. These results validate the pH-controlled behavior of the designed structure and suggest that simulative approaches can be successfully coupled with experimental data to characterize responsive DNA-based nanodevices.

Ru/Fe bimetallic complexes: Synthesis, characterization, cytotoxicity and study of their interactions with DNA/HSA and human topoisomerase IB.

Three ruthenium/iron-based compounds, 1: [Ru(MIm)(bipy)(dppf)]PF6 (MIm = 2-mercapto-1-methylimidazole anion), 2: [RuCl(Im)(bipy)(dppf)]PF6 (Im = imidazole), and 3: [Ru(tzdt)(bipy)(dppf)]PF6 (tzdt = 1,3-thiazolidine-2-thione anion) (dppf = 1,1'-bis(diphenylphosphine)ferrocene and bipy = 2,2'-bipyridine), were synthesized, and characterized by elemental analyses, conductivity, UV/Vis, IR, 1H, 13C and 31P{1H} NMR spectroscopies, and by electrochemical technique. The complex 3 was also characterized by single-crystal X-ray. The three ruthenium(II) complexes show cytotoxicity against DU-145 (prostate carcinoma cells) and A549 (lung carcinoma cells) tumor cells. The free ligands do not exhibit any cytotoxic activity, such as evident by the IC50 values higher than 200 µM. UV/Vis and viscosity experiments showed that the complexes interact weakly with the DNA molecule, via electrostatic forces. The interaction of the complexes 1-3 with the HSA is moderate, with Kb values in range of 105-107 M-1, presenting a static mechanism of interaction stabilized by hydrophobic. Complexes 2 and 3 showed high affinity for the FA7 HSA site as evidenced by fluorescence spectroscopy and molecular docking. Complexes 1-3 were tested as potential human Topoisomerase IB inhibitors by analysing the different steps of the enzyme catalytic cycle. The results indicate that all compounds efficiently inhibit the DNA relaxation and the cleavage reaction, in which the effect increases upon pre-incubation. Complexes 1 and 2 are also able to slow down the religation reaction.

A molecular dynamics simulation study decodes the early stage of the disassembly process abolishing the human SAMHD1 function.

The human sterile alpha motif SAM and HD domain-containing protein 1 (SAMHD1) restricts in non-cycling cells type the infection of a large range of retroviruses including HIV-1, reducing the intracellular pool concentration of deoxynucleoside triphosphates (dNTPs) required for the reverse transcription of the viral genome. The enzyme is in equilibrium between different forms depending on bound cofactors and substrate. In this work, two SAMHD1 three-dimensional models have been investigated through classical molecular dynamics simulation, to define the role of cofactors and metal ions in the association of the tetrameric active form. A detailed analysis of the inter-subunit interactions, taking place at the level of helix 13, indicates that removal of metal ions and cofactors induces an asymmetric loosening of the monomer-monomer interface leading to the formation of a loose tetramer where the two dimeric interfaces are weakened in different way.

SSADH deficiency in an Italian family: a novel ALDH5A1 gene mutation affecting the succinic semialdehyde substrate binding site.

SSADH deficiency (SSADHD) is a rare autosomal recessively inherited metabolic disorder. It is associated with mutations of ALDH5A1 gene, coding for the homotetrameric enzyme SSADH. This enzyme is involved in γ-aminobutyric acid (GABA) catabolism, since it oxidizes succinic semialdehyde (SSA) to succinate. Mutations in ALDH5A1 gene result in the abnormal accumulation of γ-hydroxybutyrate (GHB), which is pathognomonic of SSADHD. In the present report, diagnosis of SSADHD in a three-month-old female was achieved by detection of high levels of GHB in urine. Sequence analysis of ALDH5A1 gene showed that the patient was a compound heterozygote for c.1226G > A (p.G409D) and the novel missense mutation, c.1498G > C (p.V500 L). By ALDH5A1 gene expression in transiently transfected HEK293 cells and enzyme activity assays, we demonstrate that the p.V500 L mutation, despite being conservative, produces complete loss of enzyme activity. In silico protein modelling analysis and evaluation of tetramer destabilizing energies suggest that structural impairment and partial occlusion of the access channel to the active site affect enzyme activity. These findings add further knowledge on the missense mutations associated with SSADHD and the molecular mechanisms underlying the loss of the enzyme activity.