Metal-Complexes Bearing Releasable CO Differently Modulate Amyloid Aggregation.

Neurodegenerative diseases are often associated with an uncontrolled amyloid aggregation. Hence, many studies are oriented to discover new compounds that are able to modulate self-recognition mechanisms of proteins involved in the development of these pathologies. Herein, three metal-complexes able to release carbon monoxide (CORMs) were analyzed for their ability to affect the self-aggregation of the amyloidogenic fragment of nucleophosmin 1, corresponding to the second helix of the three-helix bundle located in the C-terminal domain of the protein, i.e., NPM1264-277, peptide. These complexes were two cymantrenes coordinated to the nucleobase adenine (Cym-Ade) and to the antibiotic ciprofloxacin (Cym-Cipro) and a Re(I)-compound containing 1,10-phenanthroline and 3-CCCH2NHCOCH2CH2-6-bromo-chromone as ligands (Re-Flavo). Thioflavin T (ThT) assay, UV-vis absorption and fluorescence spectroscopies, scanning electron microscopy (SEM), and electrospray ionization mass spectrometry (ESI-MS) indicated that the three compounds have different effects on the peptide aggregation. Cym-Ade and Cym-Cipro act as aggregating agents. Cym-Ade induces the formation of NPM1264-277 fibers longer and stiffer than that formed by NPM1264-277 alone; irradiation of complexes speeds the formation of fibers that are more flexible and thicker than those found without irradiation. Cym-Cipro induces the formation of longer fibers, although slightly thinner in diameter. Conversely, Re-Flavo acts as an antiaggregating agent. Overall, these results indicate that metal-based CORMs with diverse structural features can have a different effect on the formation of amyloid fibers. A proper choice of ligands attached to metal can allow the development of metal-based drugs with potential application as antiamyloidogenic agents.

Hydrogelation tunability of bioinspired short peptides.

Supramolecular assemblies of short peptides are experiencing a stimulating flowering. Herein, we report a novel class of bioinspired pentapeptides, not bearing Phe, that form hydrogels with fibrillar structures. The inherent sequence comes from the fragment 269-273 of nucleophosmin 1 protein, that is normally involved in liquid-liquid phase separation processes into the nucleolus. By means of rheology, spectroscopy, and scanning microscopy the crucial roles of the extremities in the modulation of the mechanical properties of hydrogels were elucidated. Three of four peptide showed a typical shear-thinning profile and a self-assembly into hierarchical nanostructures fibers and two of them resulted biocompatible in MCF7 cells. The presence of an amide group at C-terminal extremity caused the fastest aggregation and the major content of structured intermediates during gelling process. The tunable mechanical and structural features of this class of hydrogels render derived supramolecular systems versatile and suitable for future biomedical applications.

Small molecules enhancers of amyloid aggregation of C-terminal domain of Nucleophosmin 1 in acute myeloid leukemia.

The "Acute Myeloid Leukemia with gene mutations'' group includes mutations in Nucleophosmin 1(NPM1) that is an abundant multifunctional protein with chaperon functions. This protein also takes part to rRNA maturation in ribosome biogenesis, tumor suppression and nucleolar stress response. Mutations of NPM1 associated to AML present in its C-terminal domain (CTD) unable its correct folding and confer it an aberrant cytoplasmatic localization (NPMc+). AML cells with NPM1 mutations retain a certain amount of wt NPM1 in the nucleolus and since NPM1 acts as a hub protein, the nucleolus of AML cells are more vulnerable with respect to cells expressing only wt NPM1. Thus, interfering with the levels or the oligomerization status of NPM1 may influence its capability to properly build up the nucleolus in AML cells. Our biophysical recent results demonstrated that AML-CTDs contain regions prone to amyloid aggregation and, herein, we present results oriented to exploit this amylodogenesis in a potential therapeutic way. We evaluated the different ability of two small molecules to enhance amyloid aggregation through complementary biophysical approaches as fluorescence and Circular Dichroism spectroscopies, Scanning Electron Microscopy and cell-viability assays, to evaluate the cytoxicity of these molecules in AML cells lines. These findings could pave the way into molecular mechanisms of NPM1c and in novel therapeutic routes toward AML progression.

Less COVID-19 deaths in southern and insular Italy explained by forest bathing, Mediterranean environment, and antiviral plant volatile organic compounds.

The coronavirus disease 2019 (COVID-19) is causing major sanitary and socioeconomic issues, yet some locations are less impacted than others. While densely populated areas are likely to favor viral transmission, we hypothesize that other environmental factors could explain lower cases in some areas. We studied COVID-19 impact and population statistics in highly forested Mediterranean Italian regions versus some northern regions where the amount of trees per capita is much lower. We also evaluated the affinity of Mediterranean plant-emitted volatile organic compounds (VOCs) isoprene, α-pinene, linalool and limonene for COVID-19 protein targets by molecular docking modeling. Results show that while mean death number increased about 4 times from 2020 to 2021, the percentage of deaths per population (0.06-0.10%) was lower in the greener Mediterranean regions such as Sardinia, Calabria and Basilica versus northern regions with low forest coverage, such as Lombardy (0.33%) and Emilia Romagna (0.29%). Data also show that the pandemic severity cannot be explained solely by population density. Modeling reveals that plant organic compounds could bind and interfere with the complex formed by the receptor binding domain of the coronavirus spike protein with the human cell receptor. Overall, our findings are likely explained by sea proximity and mild climate, Mediterranean diet and the abundance of non-deciduous Mediterranean plants which emit immunomodulatory and antiviral compounds. Potential implications include 'forest bathing' as a therapeutic practice, designing nasal sprays containing plant volatile organic compounds, and preserving and increasing forest coverage. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10311-021-01309-5.

Old growth forests and large old trees as critical organisms connecting ecosystems and human health. A review.

Old forests containing ancient trees are essential ecosystems for life on earth. Mechanisms that happen both deep in the root systems and in the highest canopies ensure the viability of our planet. Old forests fix large quantities of atmospheric CO2, produce oxygen, create micro-climates and irreplaceable habitats, in sharp contrast to young forests and monoculture forests. The current intense logging activities induce rapid, adverse effects on our ecosystems and climate. Here we review large old trees with a focus on ecosystem preservation, climate issues, and therapeutic potential. We found that old forests continue to sequester carbon and fix nitrogen. Old trees control below-ground conditions that are essential for tree regeneration. Old forests create micro-climates that slow global warming and are irreplaceable habitats for many endangered species. Old trees produce phytochemicals with many biomedical properties. Old trees also host particular fungi with untapped medicinal potential, including the Agarikon, Fomitopsis officinalis, which is currently being tested against the coronavirus disease 2019 (COVID-19). Large old trees are an important part of our combined cultural heritage, providing people with aesthetic, symbolic, religious, and historical cues. Bringing their numerous environmental, oceanic, ecological, therapeutic, and socio-cultural benefits to the fore, and learning to appreciate old trees in a holistic manner could contribute to halting the worldwide decline of old-growth forests.

Forest-bathing and physical activity as weapons against COVID-19: a review.

Strengthening the immune system in order to better withstand the threat of COVID-19 is an important way to ensure the protection of our health against the current pandemic associated with SARS-CoV-2. There are many ways to achieve this, but with current circumstances, certain modalities stand out as being the most valid and are certainly worth greater consideration. Here we review the effects that particular immuno-strengthening activities can have on limiting the severity of COVID-19 disease as well as preventing virus infection. Physical activity, in particular, should not be discounted as an important method of prevention of viral diseases as it triggers many biological processes within the human body which in turn lead to heightened natural defences against viral infections. When exercise is performed in forested areas, these protective health benefits may be increased since many plant species emit biogenic volatile compounds (VOCs) which, when inhaled, have many protective properties. These VOCs have been shown in particular to have immunostimulatory effects on the human body and, thus, they could be of use in the prevention and/or treatment of COVID-19. Being amongst trees may also help to alleviate stress and anxiety, lowering cortisol levels and consequently helping the proper functioning of the immune system. In the following work, we have performed an analysis of the available scientific literature which looks at the effects of physical exercise as well as 'forest-bathing' on the immune system's ability to fight disease, especially of course as it relates to COVID-19. Our review aims at shedding light on the benefits of exercising outdoors in green areas and suggests reforestation as a protective measure against future outbreaks.

Supramolecular aggregation of colloidal natural organic matter masks priority pollutants released in water from peat soil.

Natural organic matter (NOM) from Sphagnum peat soil is extracted in water and subjected to several investigations to obtain structural and conformational information. Data show that the extracted NOM is self-organized in colloidal aggregates of variable sizes (from nano to micro scales, depending on the solvent composition, i.e., ultrapure water, solutions with denaturing agents, acetone, ethanol). Aggregates are formed by highly heterogeneous classes of organic compounds. According to the results of nuclear magnetic resonance and fluorescence measurements, the three-dimensional structure of aggregates, revealed by scanning electron microscope imaging, is supposed to be stabilized by the exposition of polar functional groups to the solvent, with consequent formation of hydrogen bonds, dipole-interactions and cation bridging. In contrast, the inner part of the aggregates displays hydrophobic features and is hypothesized to be further reinforced by the establishment of π-stacking interactions. The structure is assumed to be a supramolecular aggregation of small-medium oligomeric fragments (Max 750 Da) in which priority pollutants are entrapped by dispersive forces. The structures are shown to be nanosized spheroidal particles further aggregated to form higher dimension supra-structures. Carbohydrates play primary role, stabilizing the structure and giving marked hydrophilic properties to the aggregates.

Molecular Basis of the Therapeutical Potential of Clove (Syzygium aromaticum L.) and Clues to Its Anti-COVID-19 Utility.

The current COronaVIrus Disease 19 (COVID-19) pandemic caused by SARS-CoV-2 infection is enormously affecting the worldwide health and economy. In the wait for an effective global immunization, the development of a specific therapeutic protocol to treat COVID-19 patients is clearly necessary as a short-term solution of the problem. Drug repurposing and herbal medicine represent two of the most explored strategies for an anti-COVID-19 drug discovery. Clove (Syzygium aromaticum L.) is a well-known culinary spice that has been used for centuries in folk medicine in many disorders. Interestingly, traditional medicines have used clove since ancient times to treat respiratory ailments, whilst clove ingredients show antiviral and anti-inflammatory properties. Other interesting features are the clove antithrombotic, immunostimulatory, and antibacterial effects. Thus, in this review, we discuss the potential role of clove in the frame of anti-COVID-19 therapy, focusing on the antiviral, anti-inflammatory, and antithrombotic effects of clove and its molecular constituents described in the scientific literature.

Lower COVID-19 mortality in Italian forested areas suggests immunoprotection by Mediterranean plants.

The COVID-19 pandemic has induced dramatic effects on the population of the industrialized north of Italy, whereas it has not heavily affected inhabitants of the southern regions. This might be explained in part by human exposure to high levels of fine particulate matter (PM) in the air of northern Italy, thus exacerbating the mortality. Since trees mitigate air pollution by intercepting PM onto plant surfaces and bolster the human immune system by emitting bioactive volatile organic compounds (VOCs), we hypothesize a protective role of evergreen forested areas in southern Italy. We compared the mortality rate  due to COVID-19, the death number, the positivity rate and the forest coverage per capita in various Italian regions. Hectares of forest per capita and prevalence of deciduous versus evergreen forestal species were also estimated. In silico docking studies of potentially protective compounds found in Laurus nobilis L., a typical Mediterranean plant, were performed to search for potential antivirals. We found that the pandemic's severity was generally lower in southern regions, especially those with more than 0.3 hectares of forest per capita. The lowest mortality rates were found in southern Italy, mainly in regions like Molise (0.007%) and Basilicata (0.005%) where the forest per capita ratio is higher than 0.5 Ha/person. Our findings suggest that evergreen Mediterranean forests and shrubland plants could have protected the southern population by emission of immuno-modulating VOCs and provision of dietary sources of bioactive compounds. Moreover, in silico studies revealed a potential anti-COVID-19 activity in laurusides, which are unexplored glycosides from bay laurel. Overall, our results highlight the importance of nature conservation and applications to the search for natural antivirals.

Biological macromolecule binding and anticancer activity of synthetic alkyne-containing L-phenylalanine derivatives.

Herein, we described the synthesis of two L-phenylalanines α-derivatized with a terminal alkyne moiety whose structures differed by phenyl ring halogen substitution (two o-Cl in 1 vs. one p-Br in 2) and investigated their effect on biological macromolecules and living cells. We explored their interaction with quadruplex DNA (G4 DNA), using tel26 and c-myc as models, and bovine serum albumin (BSA). By CD spectroscopy, we found that 1 caused minor tel26 secondary structure changes, leading also to a slight thermal stabilization of this hybrid antiparallel/parallel G4 structure, while the c-myc parallel topology remained essentially unchanged upon 1 binding. Other CD evidences showed the ability of 1 to bind BSA, while molecular docking studies suggested that the same molecule could be housed into the hydrophobic cavity between sub-domains IIA, IIB, and IIIA of the protein. Furthermore, preliminary aggregation studies, based on concentration-dependent spectroscopic experiments, suggested the ability of 1 to aggregate forming noncovalent polymeric systems in aqueous solution. Differently from 1, the bromine-modified compound was able to bind Cu(II) ion, likely with the formation of a CuL2 complex, as found by UV spectroscopy. Finally, cell tests excluded any cytotoxic effect of both compounds toward normal cells, but showed slight antiproliferative effects of 2 on PC3 cancerous cells at 24 h, and of 1 on both T98G and MDA-MB-231 cancer cells at 48 h.

Fluorescence and Morphology of Self-Assembled Nucleobases and Their Diphenylalanine Hybrid Aggregates.

Studies carried out in recent decades have revealed that the ability to self-assemble is a widespread property among biomolecules. Small nucleic acid moieties or very short peptides are able to generate intricate assemblies endowed with remarkable structural and spectroscopic properties. Herein, the structural/spectroscopic characterization of aggregates formed by nucleobases and peptide nucleic acid (PNA)-peptide conjugates are reported. At high concentration, all studied nucleobases form aggregates characterized by previously unreported fluorescence properties. The conjugation of these bases, as PNA derivatives, to the dipeptide Phe-Phe leads to the formation of novel hybrid assemblies, which are characterized by an amyloid-like association of the monomers. Although these compounds share the same basic cross-ß motif, the nature and number of PNA units have an important impact on both the level of structural order and the intrinsic fluorescence of the self-assembled nanostructure.

Self-Assembly of PEGylated Diphenylalanines into Photoluminescent Fibrillary Aggregates.

Diphenylalanine (FF) represents one of the most studied self-assembling peptides. As a consequence of non-covalent interactions (aromatic stacking and hydrogen bonds), FF is able to generate different nanoarchitectures, proposed in the last years as innovative tools for several applications. The identification of the relationship between the chemical building block composition and the supramolecular structure of final material is the objective of intense research. Different FF analogues were synthetized and studied. At the state of art, in the high number of FF derivatives, PEGylation has not been studied yet, notwithstanding its role has been demonstrated for longer poly-phenylalanine peptides. Herein, we describe the synthesis and the supramolecular behavior of two PEGylated-FF derivatives, PEG2-FF and PEG6-FF, in which the zwitterionic FF has been derivatized at the N-terminus with two or six ethoxylic moieties, respectively. Spectroscopic methodologies (fluorescence, circular dichroism, Fourier transform infrared) allowed the identification of their secondary structure and the calculation of the critical aggregation concentration. PEGylation of the dipeptide induces a modification of the conformational organization from nanotubes with hexagonal symmetry to ß-sheet rich fibrils. This structural organization confers photoluminescence features to the supramolecular structures.

Self-Assembling of Fmoc-GC Peptide Nucleic Acid Dimers into Highly Fluorescent Aggregates.

The study of molecules that self-assemble through noncovalent interactions is one of the most attractive topics in supramolecular chemistry. The use of short peptides or modified nucleotides as building blocks for the aggregates is particularly intriguing because these are very easy to synthesize; moreover, subtle changes in the chemical structure of such building blocks may drastically affect the properties of the aggregates. The ability of peptide nucleic acids (PNA) to aggregate has been very little explored, despite its practical applications. In this work we investigated the self-assembling properties of a PNA dimer, conjugated at the N-terminus to a fluorenylmethoxycarbonyl group. This PNA dimer forms nano-aggregates at low concentration in CHCl3 /CH3 OH mixtures. The aggregates retain very interesting fluorescent properties (high quantum yield in the visible region with lifetimes on the nanosecond scale), which make them promising materials for applications in optoelectronics.

Assembly modes of hexaphenylalanine variants as function of the charge states of their terminal ends.

The ability of peptides to self-assemble represents a valuable tool for the development of biomaterials of biotechnological and/or biomedical interest. Diphenylalanine homodimer (FF) and its analogues are among the most promising systems in this field. The longest Phe-based building block hitherto characterized is pentaphenylalanine (F5). We studied the aggregation propensity and the structural/morphological features of assemblies of zwitterionic hexaphenylalanine H+-F6-O- and of three variants characterized by different charged states of the terminal ends (Ac-F6-Amide, H+-F6-Amide and Ac-F6-O-). As previously observed for PEGylated hexaphenylalanine (PEG8-F6), all F6 variants show a strong tendency to form ß-rich assemblies in which the structural motif is constituted by antiparallel ß-strands in the cross-ß framework. Extensive replica exchange molecular dynamics simulations carried out on a pairs of F6 peptides indicate that the antiparallel ß-structure of the final assemblies is likely dictated by the preferred association modes of the individual chains in the very early stages of the aggregation process. Our data suggest that even very small F6 peptides are properly pre-organized and prone to the build-up of the final assembly.

Photoluminescent Peptide-Based Nanostructures as FRET Donor for Fluorophore Dye.

A great interest has been recently generated by the discovery that peptide-based nanostructures (NSs) endowed with cross-ß structure may show interesting photoluminescent (PL) properties. It was shown that NSs formed by PEGylated hexaphenylalanine (PEG8 -F6, PEG=polyethylene glycol) are able to emit at 460 nm when excited at 370 or 410 nm. Here, the possibility to transfer the fluorescence of these PEG8 -F6-based NSs by foster resonance electron transfer (FRET) phenomenon to a fluorescent dye was explored. To achieve this aim, the 4-chloro-7-nitrobenzofurazan (NBD) dye was encapsulated in these NSs. Structural data in solution and in solid state, obtained by a variety of techniques (circular dichroism, Fourier-transform infrared spectroscopy, wide-angle X-ray scattering, and small-angle X-ray scattering), indicated that the organization of the peptide spine of PEG8 -F6 NS, which consists of anti-parallel ß-sheets separated by a dry interface made of interacting phenylalanine side chains, was maintained upon NBD encapsulation. The spectroscopic characterization of these NSs clearly showed a red-shift of the emission fluorescence peak both in solution and in solid state. This shift from 460 to 530 nm indicated that a FRET phenomenon from the peptide-based to the fluorophore-encapsulated NS occurred. FRET could also be detected in the PEG8 -F6 conjugate, in which the NBD was covalently bound to the amine of the compound. On the basis of these results, it is suggested that the red-shift of the intrinsic PL of NSs may be exploited in the bio-imaging field.

Structural Characterization of PEGylated Hexaphenylalanine Nanostructures Exhibiting Green Photoluminescence Emission.

Peptides containing aromatic residues are known to exhibit spontaneous phenomena of supramolecular organization into ordered nanostructures (NSs). In this work we studied the structural behavior and optoelectronic properties of new biocompatible materials obtained by the self-assembly of a series of hexaphenylalanines (F6) modified at the N terminus by a PEG chain of different lengths. PEG12 -F6, PEG18 -F6, and PEG24 -F6 peptides were synthesized by coupling sequentially two, three, or four units of amino-carboxy-PEG6 blocks, each one containing six oxyethylene repetitions. Changes in the length and composition of the PEG chain were found to modulate the structural organization of the phenylalanine-based nanostructures. An increase in the self-aggregation tendency was observed with longer PEG chains, whereas, independently of the PEG length, the peptide NSs display cross-ß-like secondary structures with an antiparallel ß-strand arrangement. WAXS/GIWAXS diffraction patterns indicate a progressive decrease in fiber order along the series. All the PEG-F6 derivatives present blue photoluminescent (PL) emission at 460 nm, with the adduct with the longest PEG chain (PEG24 -F6) showing an additional green emission at 530 nm.

Lac-L-TTA, a novel lactose-based amino acid-sugar conjugate for anti-metastatic applications.

Here we describe the synthesis, chromatographic purification, MS and NMR characterization of a new lactosyl-derivative, i.e. a lactosyl thiophenyl-substituted triazolyl-thione L-alanine (Lac-L-TTA). This amino acid-sugar conjugate was prepared by solution synthesis in analogy to the natural fructosyl-amino acids. Furthermore, we investigated the inhibition of PC-3 prostate cancer cell colony formation by this lactose derivative in comparison with the less polar fructose-based derivative, Fru-L-TTA. This let us to compare the properties of the artificial derivative, object of the present work, with the monosaccharide-based counterpart and to obtain a preliminary information on the influence of polarity on such biological activity. A significantly higher anticancer effect of Lac-L-TTA with respect to the fructose analogue emerged from our study suggesting that the anti-metastatic potential of fructosyl-amino acids can be enhanced by increasing the polarity of the compounds, for example by introducing disaccharide moieties in place of fructose.

Synthesis and biological evaluation of a novel Amadori compound.

Here, we report the synthesis, purification, ESI MS and NMR characterization, as well as the SEM analysis of a fructosyl thiophenyl-substituted triazolyl-thione L-alanine (denominated Fru-L-TTA). This novel fructosyl derivative was obtained by solution synthesis using the Amadori reaction, in analogy to other natural fructosyl-amino acids, and fully characterized. In particular, we report an accurate NMR/MS/SEM characterization of Fru-L-TTA alongside some biological properties, and investigated to compare the properties of the artificial derivative of this work with the natural counterparts. In particular, Fru-L-TTA shares with natural fructosyl-amino acids the possibility to inhibit the colony formation of prostate cancer cells and additionally decreases their migration.

Tuning the Functional Properties of Bitter Vetch (Vicia ervilia) Protein Films Grafted with Spermidine.

Bitter vetch protein films containing positively charged spermidine, alone or with low amounts of glycerol, showed high tensile strength that progressively decreased by increasing the plasticizer concentration. Accordingly, lower film elongation at break and higher Young's module values were detected in the presence of the polyamine without or with small amounts of glycerol. These data suggest that spermidine not only acts as a plasticizer itself by ionically interacting with proteins, but that it also facilitates glycerol-dependent reduction of the intermolecular forces along the protein chains, consequently improving the film flexibility and extensibility. Thus, spermidine may be considered not only as a primary, but also as a secondary plasticizer because of its ability to enhance glycerol plasticizing performance. Such double behavior of the polyamine was confirmed by the film permeability tests, since spermidine increased the barrier properties to gases and water vapor, while glycerol emphasized this effect at low concentrations but led to its marked reversal at high concentrations. Film microscopic images also substantiated these findings, showing more compact, cohesive, and homogeneous matrices in all spermidine-containing films.