Characterization of raloxifene as a potential pharmacological agent against SARS-CoV-2 and its variants.

The new coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic, which so far has caused over 6 million deaths in 2 years, despite new vaccines and antiviral medications. Drug repurposing, an approach for the potential application of existing pharmaceutical products to new therapeutic indications, could be an effective strategy to obtain quick answers to medical emergencies. Following a virtual screening campaign on the most relevant viral proteins, we identified the drug raloxifene, a known Selective Estrogen Receptor Modulator (SERM), as a new potential agent to treat mild-to-moderate COVID-19 patients. In this paper we report a comprehensive pharmacological characterization of raloxifene in relevant in vitro models of COVID-19, specifically in Vero E6 and Calu-3 cell lines infected with SARS-CoV-2. A large panel of the most common SARS-CoV-2 variants isolated in Europe, United Kingdom, Brazil, South Africa and India was tested to demonstrate the drug's ability in contrasting the viral cytopathic effect (CPE). Literature data support a beneficial effect by raloxifene against the viral infection due to its ability to interact with viral proteins and activate protective estrogen receptor-mediated mechanisms in the host cells. Mechanistic studies here reported confirm the significant affinity of raloxifene for the Spike protein, as predicted by in silico studies, and show that the drug treatment does not directly affect Spike/ACE2 interaction or viral internalization in infected cell lines. Interestingly, raloxifene can counteract Spike-mediated ADAM17 activation in human pulmonary cells, thus providing new insights on its mechanism of action. A clinical study in mild to moderate COVID-19 patients (NCT05172050) has been recently completed. Our contribution to evaluate raloxifene results on SARS-CoV-2 variants, and the interpretation of the mechanisms of action will be key elements to better understand the trial results, and to design new clinical studies aiming to evaluate the potential development of raloxifene in this indication.

Repurposing the estrogen receptor modulator raloxifene to treat SARS-CoV-2 infection.

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates strategies to identify prophylactic and therapeutic drug candidates to enter rapid clinical development. This is particularly true, given the uncertainty about the endurance of the immune memory induced by both previous infections or vaccines, and given the fact that the eradication of SARS-CoV-2 might be challenging to reach, given the attack rate of the virus, which would require unusually high protection by a vaccine. Here, we show how raloxifene, a selective estrogen receptor modulator with anti-inflammatory and antiviral properties, emerges as an attractive candidate entering clinical trials to test its efficacy in early-stage treatment COVID-19 patients.

Xylella fastidiosa, a new plant pathogen that threatens global farming: Ecology, molecular biology, search for remedies.

Recently, the emergence of an important alien plant pathogen in Europe was evident when the Olive Quick Decline Syndrome (OQDS), a previously unknown disease causing rapid scorching and death of the trees, invested with particular virulence a substantial portion of the vast olive wood of Southern Italy (Salento, part of the Apulia region). Early evidence indicated a connection between the OQDS and the gram-negative bacterium Xylella fastidiosa. This bacterium can target several important crops, so that researchers from all over the world have investigated its association with host plants and vectors, the molecular biology of the infection mechanism, and the molecular reaction of the infected plants. Potentially resistant or tolerant cultivars and molecular targets which might be useful to control the infection have been identified. In vitro tests of compounds active against Xylella have also been performed. In this contribution, the literature and the available data will be reviewed to provide an up-to-date picture of the currently available knowledge on the role of Xylella in OQDS, and in diseases of other plants, with focus on the emerging threats to European farming.

Automatic detection of image manipulations in the biomedical literature.

Images in scientific papers are used to support the experimental description and the discussion of the findings since several centuries. In the field of biomedical sciences, in particular, the use of images to depict laboratory results is widely diffused, at such a level that one would not err in saying that there is barely any experimental paper devoid of images to document the attained results. With the advent of software for digital image manipulation, however, even photographic reproductions of experimental results may be easily altered by researchers, leading to an increasingly high rate of scientific papers containing unreliable images. In this paper I introduce a software pipeline to detect some of the most diffuse misbehaviours, running two independent tests on a random set of papers and on the full publishing record of a single journal. The results obtained by these two tests support the feasibility of the software approach and imply an alarming level of image manipulation in the published record.

Human SOD1-G93A specific distribution evidenced in murine brain of a transgenic model for amyotrophic lateral sclerosis by MALDI imaging mass spectrometry.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease caused by the degeneration of motor neurons. The transgenic mouse model carrying the human SOD1G93A mutant gene (hSOD1G93A mouse) represents one of the most reliable and widely used model of this pathology. In the present work, the innovative technique of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was applied in the study of pathological alterations at the level of small brain regions such as facial and trigeminal nuclei, which in rodents are extremely small and would be difficult to analyze with classical proteomics approaches. Comparing slices from three mice groups (transgenic hSOD1G93A, transgenic hSOD1WT, and nontransgenic, Ntg), this technique allowed us to evidence the accumulation of hSOD1G93A in the facial and trigeminal nuclei, where it generates aggregates. This phenomenon is likely to be correlated to the degeneration observed in these regions. Moreover, a statistical analysis allowed us to highlight other proteins as differentially expressed among the three mice groups analyzed. Some of them were identified by reverse-phase HPLC fractionation of extracted proteins and mass spectrometric analysis before and after trypsin digestion. In particular, the 40S ribosomal protein S19 (RPS19) was upregulated in the parenkyma and reactive glial cells in facial nuclei of hSOD1G93A mice when compared to transgenic hSOD1WT and nontransgenic ones.

iMole, a web based image retrieval system from biomedical literature.

iMole is a platform that automatically extracts images and captions from biomedical literature. Images are tagged with terms contained in figure captions by means of a sophisticate text-mining tool. Moreover, iMole allows the user to upload directly their own images within the database and manually tag images by curated dictionary. Using iMole the researchers can develop a proper biomedical image database, storing the images extracted from paper of interest, image found on the web repositories, and their own experimental images. In order to show the functioning of the platform, we used iMole to build a 2DE database. Briefly, tagged 2DE gel images were collected and stored in a searchable 2DE gel database, available to users through an interactive web interface. Images were obtained by automatically parsing 16,608 proteomic publications, which yielded more than 16,500 images. The database can be further expanded by users with images of interest trough a manual uploading process. iMole is available with a preloaded set of 2DE gel data at http://imole.biodigitalvalley.com.

Parkinson's disease plasma biomarkers: an automated literature analysis followed by experimental validation.

Diagnosis of Parkinson's disease (PD) is currently assessed by the clinical evaluation of extrapyramidal signs. The identification of specific biomarkers would be advisable, however most studies stop at the discovery phase, with no biomarkers reaching clinical exploitation. To this purpose, we developed an automated literature analysis procedure to retrieve all the background knowledge available in public databases. The bioinformatic platform allowed us to analyze more than 51,000 scientific papers dealing with PD, containing information on 4121 proteins. Out of these, we could track back 35 PD-related proteins as present in at least two published 2-DE maps of human plasma. Then, 9 different proteins (haptoglobin, transthyretin, apolipoprotein A-1, serum amyloid P component, apolipoprotein E, complement factor H, fibrinogen γ, thrombin, complement C3) split into 32 spots were identified as a potential diagnostic pattern. Eventually, we compared the collected literature data to experimental gels from 90 subjects (45 PD patients, 45 non-neurodegenerative control subjects) to experimentally verify their potential as plasma biomarkers of PD.

Synthesis of a diaminopropanoic acid-based nucleoamino acid and assembly of cationic nucleopeptides for biomedical applications.

In this work, we report a synthetic approach to a Fmoc-protected nucleoamino acid, based on L-diaminopropanoic acid, carrying the DNA nucleobase on the alpha-amino group by means of an amide bond, suitable for the solid-phase synthesis of novel nucleopeptides of potential interest in biomedicine. After ESI-MS and NMR characterization this building block was used for the assembly of a thymine-functionalized nucleopeptide, composed of nucleobase-containing L-diaminopropanoic acid moieties and underivatized L-lysine residues alternated in the backbone. Circular dichroism studies performed on the cationic nucleopeptide and adenine-containing DNA and RNA molecules suggested that the thymine-containing peptide is able to interact with both DNA and RNA. In particular, a significant conformational variation in the RNA structure was suggested by CD studies. Human serum stability assays were also conducted on the cationic nucleopeptide, which was found to be highly resistant to enzymatic degradation.

Dakin-West reaction on 1-thyminyl acetic acid for the synthesis of 1,3-bis(1-thyminyl)-2-propanone, a heteroaromatic compound with nucleopeptide-binding properties.

This work deals with the Dakin-West synthesis, starting from the nucleoamino acid 1-thyminyl acetic acid, as well the NMR, ESI MS, and X-ray characterization of a heteroaromatic compound denominated by us T(2)CO, comprising two thymine moieties anchored to a 2-propanonic unit, the spectroscopic properties of which were studied by UV as a function of temperature and ionic strength. Preliminary binding-studies with molecules of biomedical interest such as nucleic acids and proteins, performed on samples containing T(2)CO, suggested that this molecule is able to interact very weakly with double-stranded RNA, whereas it does not seem to bind other nucleic acids or proteins. Moreover, by studies with fresh human serum we found that T(2)CO is resistant to enzymatic degradation till 24 h, whereas UV metal binding-studies, performed using solutions of copper (II) chloride dihydrate and nickel (II) chloride hexahydrate, revealed a certain ability of T(2)CO to bind copper (II) cation. Finally, by CD spectroscopy we investigated the influence of T(2)CO on the already described supramolecular networks based on L-serine-containing nucleopeptides. More particularly, we found that T(2)CO is able to increase the level of structuration of the non-covalent supramolecular assembly of the chiral nucleopeptides, which is a feature of remarkable interest for the development of innovative drug delivery tools.

G-quadruplex-forming oligonucleotide conjugated to magnetic nanoparticles: synthesis, characterization, and enzymatic stability assays.

In the present work, we report the conjugation of superparamagnetic nanoparticles to a fluorescently labeled oligodeoxyribonucleotide (ODN) able to fold into stable unimolecular guanine quadruple helix under proper ion conditions by means of its thrombin-binding aptamer (TBA) sequence. The novel modified ODN, which contained a fluorescent dU(Py) unit at 3'-end and a 12-amino-dodecyl spacer (C(12)-NH(2)) at 5' terminus, was characterized by ESI-MS and optical spectroscopy (UV, CD, fluorescence), and analyzed by RP-HPLC chromatography and electrophoresis. From CD and fluorescence experiments, we verified that dU(Py) and C(12)-NH(2) incorporation does not interfere with the conformational stability of the G-quadruplex. Subsequently, the conjugation of the pyrene-labeled ODN with the magnetite particles was performed, and the ODN-conjugated nanoparticles were studied through optical spectroscopy (UV, CD, fluorescence) and by enzymatic and chemical assays. We found that the nanoparticles enhanced the stability of the TBA ODN to enzymatic degradation. Finally, we evaluated the amount of the TBA-conjugated nanoparticles immobilized on a magnetic separator in view of the potential use of the nanosystem for the magnetic capture of thrombin from complex mixtures.

Synthesis and aggregation properties of a novel enzymatically resistant nucleoamino acid.

In this work, we describe the synthesis, evaluation of some biological properties, such as DNA- and RNA-binding ability and in sero stability, as well as the supramolecular assembly of a novel nucleoamino acid based on L-spinacine. More particularly, a thymine-containing L-spinacine derivative was synthesized in liquid phase by a simple peptide-coupling procedure. Subsequently, nucleic acid and Cu(2+)-binding ability, as well as self-assembly properties of the novel nucleoamino acid, were investigated by spectroscopy (CD and UV) and laser light scattering which furnished interesting information on the assembly of supramolecular networks based on the peptidyl nucleoside analog. Finally, nucleoamino acid enzymatic stability was studied and a half life of about 7 days was found in the presence of fresh human serum.

A novel Gaussian extrapolation approach for 2D gel electrophoresis saturated protein spots.

Analysis of images obtained from two-dimensional gel electrophoresis (2D-GE) is a topic of utmost importance in bioinformatics research, since commercial and academic software available currently has proven to be neither completely effective nor fully automatic, often requiring manual revision and refinement of computer generated matches. In this work, we present an effective technique for the detection and the reconstruction of over-saturated protein spots. Firstly, the algorithm reveals overexposed areas, where spots may be truncated, and plateau regions caused by smeared and overlapping spots. Next, it reconstructs the correct distribution of pixel values in these overexposed areas and plateau regions, using a two-dimensional least-squares fitting based on a generalized Gaussian distribution. Pixel correction in saturated and smeared spots allows more accurate quantification, providing more reliable image analysis results. The method is validated for processing highly exposed 2D-GE images, comparing reconstructed spots with the corresponding non-saturated image, demonstrating that the algorithm enables correct spot quantification.

Synthesis and characterization of a novel ester-based nucleoamino acid for the assembly of aromatic nucleopeptides for biomedical applications.

In this work, we report a technological approach to a novel Fmoc-protected nucleoamino acid, based on l-tyrosine, carrying the DNA nucleobase on the hydroxyl group by means of an ester bond, suitable for the solid-phase synthesis of novel aromatic nucleopeptides of potential interest in biomedicine. After ESI-MS and NMR characterization this building block was used for the assembly of a thymine-functionalized tetrapeptide, composed of nucleobase-containing and underivatized l-tyrosine moieties alternated in the backbone.

DNA-based strategies for blocking HMGB1 cytokine activity: design, synthesis and preliminary in vitro/in vivo assays of DNA and DNA-like duplexes.

In this work we report the design and synthesis of kinked oligonucleotide duplexes as potential inhibitors of HMGB1, a cytokine which triggers a broad range of immunological effects. We found that the designed ligands can interact with HMGB1, as evidenced by circular dichroism spectroscopy, and are able to block some extracellular effects induced by the protein, such as cellular proliferation and migration, as we demonstrated by in vitro biological assays. After selecting the most stable and active kinked duplex, we synthesized the corresponding PNA/DNA chimeric duplex which resulted to be more resistant to enzymatic degradation, and showed a biological activity comparable to that of the natural duplex. Preliminary in vivo assays in a mouse inflammatory model, showed a significant decrease of the mortality after administration of the PNA/DNA kinked duplex to LPS-treated mice.

Synthesis, biological evaluation and supramolecular assembly of novel analogues of peptidyl nucleosides.

This work concerns the synthesis, the supramolecular assembly and the evaluation of some biological properties, such as DNA and RNA-binding ability and human serum stability, of novel nucleopeptides. These compounds are of potential interest for the well-known properties that similar compounds, such as natural peptidyl nucleosides, possess in biology and medicine and also for the possibility to realize nucleopeptide-based supramolecular systems useful for drug and gene delivery applications. More particularly, all four nucleobase-containing peptides were synthesized by solid phase synthesis, purified by HPLC and characterized by NMR and ESI-MS. Subsequently, nucleopeptide self-assembly as well as DNA and RNA-binding ability were investigated by CD spectroscopy and further information on the formation of molecular networks, based on the peptidyl nucleoside analogues and nucleic acids, was obtained by Laser Light Scattering. Finally, nucleopeptide enzymatic stability was studied and a half life of about 2 hours was found in the presence of 50% fresh human serum.

RNA-binding and viral reverse transcriptase inhibitory activity of a novel cationic diamino acid-based peptide.

A novel cationic peptide based on L-lysine and L-diaminobutyric acid was prepared for the first time by solid phase synthesis. After HPLC purification and ESI MS characterization, we studied by CD and IR spectroscopy the structural features of the novel basic peptide, which is able to form a ß-turn-like structure. Furthermore, its interaction with DNA and RNA was investigated by CD and UV spectroscopy, which revealed a preferential RNA-binding ability of the sequential peptide, whereas its inhibitory activity toward HIV and Moloney murine leukemia virus (MMLV) reverse transcriptase action was evaluated by semiquantitative PCR. The cationic sequential peptide was able to inhibit the reverse transcriptase activity in both cases, even if our PCR data suggested a major activity in the case of HIV-RT, probably due to the stronger cationic peptide-protein interaction evidenced by UV spectroscopy.