Experimental and in silico evaluations of the possible molecular interaction between airborne particulate matter and SARS-CoV-2.

During the early stage of the COVID-19 pandemic (winter 2020), the northern part of Italy has been significantly affected by viral infection compared to the rest of the country leading the scientific community to hypothesize that airborne particulate matter (PM) could act as a carrier for the SARS-CoV-2. To address this controversial issue, we first verified and demonstrated the presence of SARS-CoV-2 RNA genome on PM2.5 samples, collected in the city of Bologna (Northern Italy) in winter 2021. Then, we employed classical molecular dynamics (MD) simulations to investigate the possible recognition mechanism(s) between a newly modelled PM2.5 fragment and the SARS-CoV-2 Spike protein. The potential molecular interaction highlighted by MD simulations suggests that the glycans covering the upper Spike protein regions would mediate the direct contact with the PM2.5 carbon core surface, while a cloud of organic and inorganic PM2.5 components surround the glycoprotein with a network of non-bonded interactions resulting in up to 4769 total contacts. Moreover, a binding free energy of -207.2 ± 3.9 kcal/mol was calculated for the PM-Spike interface through the MM/GBSA method, and structural analyses also suggested that PM attachment does not alter the protein conformational dynamics. Although the association between the PM and SARS-CoV-2 appears plausible, this simulation does not assess whether these established interactions are sufficiently stable to carry the virus in the atmosphere, or whether the virion retains its infectiousness after the transport. While these key aspects should be verified by further experimental analyses, for the first time, this pioneering study gains insights into the molecular interactions between PM and SARS-CoV-2 Spike protein and will support further research aiming at clarifying the possible relationship between PM abundance and the airborne diffusion of viruses.

Peptide-Functionalized and Drug-Loaded Tomato Bushy Stunt Virus Nanoparticles Counteract Tumor Growth in a Mouse Model of Shh-Dependent Medulloblastoma.

Sonic hedgehog medulloblastoma (SHH-MB) accounts for 25-30% of all MBs, and conventional therapy results in severe long-term side effects. New targeted therapeutic approaches are urgently needed, drawing also on the fields of nanoparticles (NPs). Among these, plant viruses are very promising, and we previously demonstrated that tomato bushy stunt virus (TBSV), functionalized on the surface with CooP peptide, specifically targets MB cells. Here, we tested the hypothesis that TBSV-CooP can specifically deliver a conventional chemotherapeutic drug (i.e., doxorubicin, DOX) to MB in vivo. To this aim, a preclinical study was designed to verify, by histological and molecular methods, if multiple doses of DOX-TBSV-CooP were able to inhibit tumor progression of MB pre-neoplastic lesions, and if a single dose was able to modulate pro-apoptotic/anti-proliferative molecular signaling in full-blown MBs. Our results demonstrate that when DOX is encapsulated in TBSV-CooP, its effects on cell proliferation and cell death are similar to those obtained with a five-fold higher dose of non-encapsulated DOX, both in early and late MB stages. In conclusion, these results confirm that CooP-functionalized TBSV NPs are efficient carriers for the targeted delivery of therapeutics to brain tumors.

The interaction of DNMT1 and DNMT3A epigenetic enzymes with phthalates and perfluoroalkyl substances: an in silico approach.

The occurrence of long-lasting adverse effects of the environmental contaminants on human health is a current emerging issue. In particular, phthalates, poly- and perfluoroalkyl substances are proposed to trigger toxic effects as well as persistent changes on human development and metabolism by different mechanisms, including epigenetic modifications, although the specific underlying pathways are still unknown. This study contributes to identify the potential molecular initiating events of epigenetic-mediated adverse effects by an in silico approach, which combines molecular docking and molecular dynamics simulation. The approach probes the potential molecular interaction between several different phthalates and persistent organic pollutants and a specific class of epigenetic modulators, namely the DNA methyltransferases (DNMTs). The dynamics of interaction and the binding free energies of the ligand-DNMTs complexes demonstrated that pollutants can be classified into two main groups, according to the ligand-target complex stability: (1) a larger class of phthalates (DBP, DEHP, MBP and MEHP) acting as inhibitors of the enzymatic activity of the epigenetic targets and (2) a smaller class of phthalates (DMP and MMP) and perfluoroalkyl substances (PFOA and PFOS) which do not interact stably with the human DNMTs. These findings provide the first valuable in silico insights on the ability of these specific environmental pollutants to directly bind and inhibit a key class of epigenetic regulators. Communicated by Ramaswamy H. Sarma.

Tomato Bushy Stunt Virus Nanoparticles as a Platform for Drug Delivery to Shh-Dependent Medulloblastoma.

Medulloblastoma (MB) is a primary central nervous system tumor affecting mainly young children. New strategies of drug delivery are urgent to treat MB and, in particular, the SHH-dependent subtype-the most common in infants-in whom radiotherapy is precluded due to the severe neurological side effects. Plant virus nanoparticles (NPs) represent an innovative solution for this challenge. Tomato bushy stunt virus (TBSV) was functionally characterized as a carrier for drug targeted delivery to a murine model of Shh-MB. The TBSV NPs surface was genetically engineered with peptides for brain cancer cell targeting, and the modified particles were produced on a large scale using Nicotiana benthamiana plants. Tests on primary cultures of Shh-MB cells allowed us to define the most efficient peptides able to induce specific uptake of TBSV. Immunofluorescence and molecular dynamics simulations supported the hypothesis that the specific targeting of the NPs was mediated by the interaction of the peptides with their natural partners and reinforced by the presentation in association with the virus. In vitro experiments demonstrated that the delivery of Doxorubicin through the chimeric TBSV allowed reducing the dose of the chemotherapeutic agent necessary to induce a significant decrease in tumor cells viability. Moreover, the systemic administration of TBSV NPs in MB symptomatic mice, independently of sex, confirmed the ability of the virus to reach the tumor in a specific manner. A significant advantage in the recognition of the target appeared when TBSV NPs were functionalized with the CooP peptide. Overall, these results open new perspectives for the use of TBSV as a vehicle for the targeted delivery of chemotherapeutics to MB in order to reduce early and late toxicity.

Characterization Of Blood-Brain Barrier Crossing And Tumor Homing Peptides By Molecular Dynamics Simulations.

INTRODUCTION: The new frontier of tumor diagnosis and treatment relies on the development of delivery strategies capable of allowing the specific targeting of the diagnostic agents/chemotherapeutics, avoiding side effects. In the case of brain tumors, achieving this goal is made more difficult by the presence of the blood-brain barrier (BBB). Peptides have been revealed as excellent candidates for both BBB crossing and specific cancer homing. Nanoparticles (NPs), functionalized with BBB crossing and tumor homing (TH) peptides, are emerging as smart theranostic systems. However, there is still poor knowledge concerning the molecular structure and dynamical properties of these peptides, essential requirements for a suitable functionalization of the delivery systems themselves. METHODS: In this work, by means of molecular dynamics (MD) simulations, we have extensively characterized the structural and dynamical behavior of several peptides, known to be endowed of BBB crossing and TH properties. RESULTS: The simulations point out that, on the basis of their conformational dynamics, the peptides can be classified in two main groups: 1) peptides assuming a specific structural conformation, a feature that could be important for interacting with the molecular target but that may limit their use as functionalizing molecules and 2) highly flexible peptides whose interaction with the target may be independent of a particular structural conformation and that may represent good candidates for the functionalization of theranostic NP-based platforms. DISCUSSION: Such findings may be useful for the de novo designing of NP-based delivery systems.

Dynamics at a Peptide-TiO2 Anatase (101) Interface.

The interface between biological matter and inorganic materials is a widely investigated research topic due to possible applications in biomedicine and nanotechnology. In this context, the molecular level adsorption mechanism that drives specific recognition between small peptide sequences and inorganic surfaces represents an important topic likely to provide much information useful for designing bioderived materials. Here, we investigate the dynamics at the interface between a Ti-binding peptide sequence (AMRKLPDAPGMHC) and a TiO2 anatase surface by using molecular dynamics (MD) simulations. In the simulations the adsorption mechanism is characterized by diffusion of the peptide from the bulk water phase toward the TiO2 surface, followed by the anchoring of the peptide to the surface. The anchoring is mediated by the interfacial water layers by means of the charged groups of the side chains of the peptide. The peptide samples anchored and dissociated states from the surface and its conformation is not affected by the surface when anchored.

Adsorption of Modified Arg, Lys, Asp, and Gln to Dry and Hydrated ZnO Surface: A Density Functional Theory Study.

The interface of biological molecules with inorganic surfaces has been the subject of several recent studies. Experimentally some amino acids are evidenced to play a critical role in the adhesion and selectivity on oxide surfaces; however, detailed information on how the water molecules on the hydrated surface are able to mediate the adsorption is still missing. Accurate total energy ab initio calculations based on dispersion-corrected density functional theory have been performed to investigate the adsorption of selected amino acids on the hydrated ZnO(101̅0) surface, and the results are presented and discussed in this paper. We have also investigated the role played by water in the determination of the most energetically favorable adsorption configurations of the selected amino acids. We have found that for some amino acids the most energetically favorable configurations involve the deprotonation of the molecule if the water screening is not effective.

Water driven adsorption of amino acids on the (101) anatase TiO2 surface: an ab initio study.

Arg, Lys and Asp amino acids are known to play a critical role in the adhesion of the RKLPDA engineered peptide on the (101) surface of the titania anatase phase. To understand their contribution to peptide adhesion, we have considered the relevant charge states due to protonation (Arg and Lys) or deprotonation (Asp) occurring in neutral water solution, and studied their adsorption on the (101) anatase TiO2 surface by ab initio total energy calculations based on density functional theory. The adsorption configurations on the hydrated surface are compared to those on the dry surface considering also the presence of the hydration shell around amino acid side-chains. This study explains how water molecules mediate the adsorption of charged amino acids showing that protonated amino acids are chemically adsorbed much more strongly than de-protonated Asp. Moreover it is shown that the polar screening of the hydration shell reduces the adsorption energy of the protonated amino acids to a small extent, thus evidencing that both Arg and Lys strongly adhere on the (101) anatase TiO2 surface in neutral water solution and that they play a major role in the adhesion of the RKLPDA peptide.

Structure-based design and experimental engineering of a plant virus nanoparticle for the presentation of immunogenic epitopes and as a drug carrier.

Biomaterials research for the discovery of new generation nanoparticles is one of the most active areas of nanotechnology. In the search of nature-made nanometer-sized objects, plant virus particles appear as symmetrically defined entities that can be formed by protein self-assembly. In particular, in the field of plant virology, there is plenty of literature available describing the exploitation of plant viral cages to produce safe vaccine vehicles and nanoparticles for drug delivery. In this context, we have investigated on the use of the artichoke mottled crinkle virus (AMCV) capsid both as a carrier of immunogenic epitopes and for the delivery of anticancer molecules. A dual approach that combines both in silico tools and experimental virology was applied for the rational design of immunologically active chimeric virus-like particles (VLPs) carrying immunogenic peptides. The atomic structures of wild type (wt) and chimeric VLPs were obtained by homology modeling. The effects of insertion of the HIV-1 2F5 neutralizing epitope on the structural stability of chimeric VLPs were predicted and assessed by detailed inspection of the nanoparticle intersubunit interactions at atomic level. Wt and chimeric VLPs, exposing on their surface the 2F5 epitope, were successfully produced in plants. In addition, we demonstrated that AMCV capsids could also function as drug delivery vehicles able to load the chemotherapeutic drug doxorubicin. To our knowledge, this is the first systematic predictive and empirical research addressing the question of how this icosahedral virus can be used for the production of both VLPs and viral nanoparticles for biomedical applications.

Variability and expression profile of the DRF1 gene in four cultivars of durum wheat and one triticale under moderate water stress conditions.

The dehydration responsive element binding (DREB) proteins are important transcription factors that contribute to stress endurance in plants triggering the expression of a set of abiotic stress-related genes. A DREB2-related gene, previously referred to as dehydration responsive factor 1 (DRF1) was originally isolated and characterized in durum wheat. The aim of this study was to monitor the expression profiles of three alternatively spliced TdDRF1 transcripts during dehydration experiments and to evaluate the effects of genetic diversity on the molecular response, using experimental conditions reflecting as closely as possible water stress perceived by cereals in open field. To investigate the effect of moderate water stress conditions, time-course dehydration experiments were carried out under controlled conditions in the greenhouse on four durum wheat and one triticale genotypes. Differences were observed in molecular patterns, thus, suggesting a genotype dependency of the DRF1 gene expression in response to the stress induced. The biodiversity of the transcripts of the DRF1 gene was explored in order to assess the level of polymorphism and its possible effects on structure and function of putative proteins. A total of nine haplotypes were identified in the sequences cloned, seven of which encompassing polymorphisms in exon 4, including the region codifying for the DNA binding Apetala2 (AP2) domain. The 3D structural models of the AP2 domain were generated by homology modelling using the variability observed. The polymorphisms analysed did not significantly affect the structural arrangement of the DNA binding domains, thus resulting compatible with the putative functionality.

Bacterial cytoplasm production of an EGFP-labeled single-chain Fv antibody specific for the HER2 human receptor.

The human epidermal growth factor receptor 2 (HER2) is the main diagnostic marker of breast and ovary cancers. Here, to obtain a rapid and sensitive immunodiagnostic tool a single-chain antibody (scFv800E6) specific for the HER2 was fused to the N-terminus of the enhanced green fluorescent protein (EGFP) by a flexible linker. The soluble production of the novel scFv800E6-EGFP protein in the cytoplasm of Escherichia coli was investigated at different induction temperatures (25, 30 and 37°C); the intrinsic fluorescent properties and the binding activity to HER2 positive tumour cells of the fusion protein were analysed. Western blotting and fluorescence analysis of SDS-PAGE revealed the presence of two scFv800E6-EGFP forms, with different mobility and optical properties, their ratio depending on the induction temperature. The fluorescent form maintained the optical fluorescence properties of EGFP and exhibited a binding activity to the HER2-expressing cells comparable to that of the non-fused scFv800E6. In addition, to provide an insight into the effect of the induction temperature on the molecular structure, the folding of the fusion protein was assessed at atomic level by performing molecular dynamics simulations of the homology-derived model of scFv800E6-EGFP at 300 K and 310 K. The comparison of the data collected at these two temperatures revealed that the higher temperature affects specific structural elements. To improve the production of the soluble and functional scFv800E6-EGFP protein, "in silico" results could be utilised for ad hoc design of the molecular structure.

Structure and dynamics of the anti-AMCV scFv(F8): effects of selected mutations on the antigen combining site.

The recombinant antibody fragment scFv(F8), which recognizes the coat protein of the plant virus AMCV, is characterized by peculiar high in vitro stability and functional folding even in reducing environments, making it fit for designing stable antibodies with desired properties. Mutagenesis and functional analysis evidenced two residues, at positions 47 and 58 of the V(H) chain, playing a crucial role in the antigen binding recognition. Here, we used a computational procedure to assess the effects of these mutations on the stability, structure and dynamics of the antigen-binding site. Structural models of the wild type scFv(F8) and of its H47 and H58 mutants were built by homology modelling and assessed by multiple 15.5ns of molecular dynamics simulations. Computational results indicate that the 47H substitution strongly affects the CDR-H(2) conformation, destabilizes the V(H)/V(L) interface and confers high conformational flexibility to the antigen-binding site, leading the mutant to functional loss. The mutation at position H58 strenghtens the binding site, bestowing a high antigen specificity on the mutant. The essential dynamics and the analysis of the protein-solvent interface further corroborate the correspondence between the extent of the structurally-determined flexibility of the binding site with the different functional behaviours proved by the wild-type and its mutants. These results may have useful implications for structure-based design of antibody combining site.

Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies.

We report on the development of the F64L/S65T/T203Y/L231H GFP mutant (E2GFP) as an effective ratiometric pH indicator for intracellular studies. E2GFP shows two distinct spectral forms that are convertible upon pH changes both in excitation and in emission with pK close to 7.0. The excitation of the protein at 488 and 458 nm represents the best choice in terms of signal dynamic range and ratiometric deviation from the thermodynamic pK. This makes E2GFP ideally suited for imaging setups equipped with the most widespread light sources and filter settings. We used E2GFP to determine the average intracellular pH (pH(i)) and spatial pH(i) maps in two different cell lines, CHO and U-2 OS, under physiological conditions. In CHO, we monitored the evolution of the pH(i) during mitosis. We also showed the possibility to target specific subcellular compartments such as nucleoli (by fusing E2GFP with the transactivator protein of HIV, (Tat) and nuclear promyelocytic leukemia bodies (by coexpression of promyelocytic leukemia protein).

Subnuclear distribution of the largest subunit of the human origin recognition complex during the cell cycle.

In eukaryotes, initiation of DNA replication requires the activity of the origin recognition complex (ORC). The largest subunit of this complex, Orc1p, has a critical role in this activity. Here we have studied the subnuclear distribution of the overexpressed human Orc1p during the cell cycle. Orc1p is progressively degraded during S-phase according to a spatio-temporal program and it never colocalizes with replication factories. Orc1p is resynthesized in G1. In early G1, the protein is distributed throughout the cell nucleus, but successively it preferentially associates with heterochromatin. This association requires a functional ATP binding site and a protein region partially overlapping the bromo-adjacent homology domain at the N-terminus of Orc1p. The same N-terminal region mediates the in vitro interaction with heterochromatin protein 1 (HP1). Fluorescence resonance energy transfer (FRET) experiments demonstrate the interaction of human Orc1p and HP1 in vivo. Our data suggest a role of HP1 in the recruitment but not in the stable association of Orc1p with heterochromatin. Indeed, the subnuclear distribution of Orc1p is not affected by treatments that trigger the dispersal of HP1.

Caveolae-mediated internalization of extracellular HIV-1 tat fusion proteins visualized in real time.

The Tat protein from HIV-1, when fused with heterologous proteins or peptides, can traverse cell membranes. This ability has generated great interest due to potential therapeutic applications. However, the relevant cellular pathway and its dynamics have not been elucidated yet. Here we unravel the intracellular fate of exogenously added Tat fused with green fluorescent protein (GFP) in live HeLa and CHO cells, from the early interaction with the plasma membrane up to the long-term accumulation in the perinuclear region. We demonstrate that the internalization process of full-length Tat and of heterologous proteins fused to the transduction domain of Tat exploits a caveolar-mediated pathway and is inhibited at 4 degrees C. Remarkably, a slow linear movement toward the nucleus of individual GFP-tagged Tat-filled caveolae with an average velocity of 3 micro m/h was observed. No fluorescence was observed in the nucleus, possibly suggesting that Tat fusion protein unfolding is required for nuclear translocation. In addition, early sensitivity to cytochalasin-D treatment indicates the essential role of the actin cytoskeleton in the displacement of Tat vesicles toward the nucleus. Our results imply that HIV-1 Tat mediates the internalization of protein cargos in a slow and temperature-dependent manner by exploiting the caveolar pathway.

Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins.

The transactivator protein of human immunodeficiency virus type 1 Tat has the unique property of mediating the delivery of large protein cargoes into the cells when present in the extracellular milieu. Here we show that Tat fusion proteins are internalized by the cells through a temperature-dependent endocytic pathway that originates from cell membrane lipid rafts and follows caveolar endocytosis. These conclusions are supported by the study of the slow kinetics of the internalization of Tat endosomes, by their resistance to nonionic detergents, the colocalization of internalized Tat with markers of caveolar endocytosis, and the impairment of the internalization process by drugs that disrupt lipid rafts or disturb caveolar trafficking. These results are of interest for all those who exploit Tat as a vehicle for transcellular protein delivery.