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Coronaviruses are highly transmissible and pathogenic viruses for humans and animals. The vast quantity of information collected about SARS-CoV-2 during the pandemic helped to unveil details of the mechanisms behind the infection, which are still largely elusive. Recent research demonstrated that different class I/II human leukocyte antigen (HLA) alleles might define an individual susceptibility to SARS-CoV-2 spreading, contributing to the differences in the distribution of the infection through different populations; additional studies suggested that the homolog of the HLA in cats, the feline leukocyte antigen (FLA), plays a pivotal role in the transmission of viruses. With these premises, this study aimed to exploit a bioinformatic approach for the prediction of the transmissibility potential of two distinct feline coronaviruses (FCoVs) in domestic cats (feline enteric coronavirus (FeCV) and feline infectious peritonitis virus (FIPV)) using SARS-CoV-2 as the reference model. We performed an epitope mapping of nonapeptides deriving from SARS-CoV-2, FeCV, and FIPV glycoproteins and predicted their affinities for different alleles included in the three main loci in class I FLAs (E, H, and K). The predicted complexes with the most promising affinities were then subjected to molecular docking and molecular dynamics simulations to provide insights into the stability and binding energies in the cleft. Results showed the FLA proteins encoded by alleles in the FLA-I H (H*00501 and H*00401) and E (E*01001 and E*00701) loci are largely responsive to several epitopes deriving from replicase and spike proteins of the analyzed coronaviruses. The analysis of the most affine epitope sequences resulting from the prediction can stimulate the development of anti-FCoV immunomodulatory strategies based on peptide drugs.

Antibodies are the macromolecules of choice to ensure specific recognition of biomarkers in biological assays. However, they present a range of shortfalls including a relatively high production cost and limited tissue penetration. Peptides are relatively small molecules able to reproduce sequences of highly specific paratopes and, although they have less biospecificity than antibodies, they offer advantages like ease of synthesis, modifications of their amino acid sequences and tagging with fluorophores and other molecules required for detection. This work presents a strategy to design peptide sequences able to recognize the CD44 hyaluronic acid receptor present in the plasmalemma of a range of cells including human bone marrow stromal mesenchymal cells. The protocol of identification of the optimal amino acid sequence was based on the combination of rational design and in silico methodologies. This protocol led to the identification of two peptide sequences which were synthesized and tested on human bone marrow mesenchymal stromal cells (hBM-MSCs) for their ability to ensure specific binding to the CD44 receptor. Of the two peptides, one binds CD44 with sensitivity and selectivity, thus proving its potential to be used as a suitable alternative to this antibody in conventional immunostaining. In the context of regenerative medicine, the availability of this peptide could be harnessed to functionalize tissue engineering scaffolds to anchor stem cells as well as to be integrated into systems such as cell sorters to efficiently isolate MSCs from biological samples including various cell subpopulations. The data here reported can represent a model for developing peptide sequences able to recognize hBM-MSCs and other types of cells and for their integration in a range of biomedical applications.

Calcium (Ca) represents about 40% of the total mineral mass, mainly in the bone, providing mechanical strength to the skeleton and teeth. An adequate Ca intake is necessary for bone growth and development in children and adolescents and for maintaining bone mineral loss in elderly age. Ca deficiency predisposes to osteopenia and osteoporosis. Healthy nutrition, including an adequate intake of Ca-rich food, is paramount to prevent and cure osteoporosis. Recently, several clinical studies have demonstrated that, in conditions of Ca dysmetabolism, Ca-rich mineral water is beneficial as a valuable source of Ca to be used as an alternative to caloric Ca-rich dairy products. Although promising, these data have been collected from small groups of participants. Moreover, they mainly regard the effect of Ca-rich mineral water on bone metabolism. In contrast, an investigation of the effect of Ca supplementation on systemic metabolism is needed to address the spreading of systemic metabolic dysfunction often associated with Ca dysmetabolism. In the present study, we analyzed urine and blood sera of 120 women in perimenopausal condition who were subjected for six months to 2l daily consumption of bicarbonate–calcium mineral water marketed under ®Lete. Remarkably, this water, in addition to being rich in calcium and bicarbonate, is also low in sodium. A complete set of laboratory tests was carried out to investigate whether the specific water composition was such to confirm the known therapeutic effects on bone metabolism. Second, but not least, urine and blood sera were analyzed using NMR-based metabolomic procedures to investigate, other than the action on Ca metabolism, potential system-wide metabolic effects. Our data show that Lete water is a valid supplement for compensating for Ca dysmetabolism and preserving bone health and integrity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus identified as the cause of the coronavirus outbreak in December 2019 (COVID-19). Like all the RNA viruses, SARS-CoV-2 constantly evolves through mutations in its genome, accumulating 1–2 nucleotide changes every month, giving the virus a selective advantage through enhanced transmissibility, greater pathogenicity, and the possibility of circumventing immunity previously acquired by an individual either by natural infection or by vaccination. Several SARS-CoV-2 variants of concern (VoC) have been identified, among which we find Alpha (Lineage B.1.1.7), Beta (Lineage B.1.351), and Gamma (Lineage P.1) variants. Most of the mutations occur in the spike (S) protein, a surface glycoprotein that plays a crucial role in viral infection; the S protein binds the host cell receptor, the angiotensin-converting enzyme of type 2 (ACE2) via the receptor binding domain (RBD) and catalyzes the fusion of the viral membrane with the host cell. In this work, we present the development of a simplified system that would afford to study the change in the SARS-CoV-2 S RBD/ACE2 binding related to the frequent mutations. In particular, we synthesized and studied the structure of short amino acid sequences, mimicking the two proteins’ critical portions. Variations in the residues were easily managed through the one-point alteration of the sequences. Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopies provide insights into ACE2 and SARS-CoV-2 S RBD structure with its related three variants (Alpha, Beta, and Gamma). Spectroscopy data supported by molecular dynamics lead to the description of an ACE2/RBD binding model in which the effect of a single amino acid mutation in changing the binding of S protein to the ACE2 receptor is predictable.

Alzheimer’s disease is a neurodegenerative disease characterized by the formation of amyloid plaques constituted prevalently by amyloid peptides. Due to the well-known challenges related to the study in solution of these peptides, several membrane-mimicking systems such as micelle constituted by detergent—i.e., DPC and SDS—have been deeply investigated. Additionally, the strategy of studying short fragments instead of the full-length peptide turned out to be advantageous in exploring the structural properties of the different moieties in Aβ in order to reproduce its pathologic effects. Several studies reveal that among Aβ fragments, Aβ(25−35) is the shortest fragment able to reproduce the aggregation process. To enrich the structural data currently available, in the present work we decided to evaluate the conformational changes adopted by Aβ(25−35) in SDS combining CD and NMR spectroscopies at different times. From the solved structures, it emerges that Aβ(25−35) passes from an unordered conformation at the time of the constitution of the system to a more ordered and energetically favorable secondary structure at day 7, which is kept for 2 weeks. These preliminary data suggest that a relatively long time affects the kinetic in the aggregation process of Aβ(25−35) in a micellar system, favoring the stabilization and the formation of a soluble helix conformation.

Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative pathology of the upper or lower motor neuron. Evaluation of ALS progression is based on clinical outcomes considering the impairment of body sites. ALS has been extensively investigated in the pathogenetic mechanisms and the clinical profile; however, no molecular biomarkers are used as diagnostic criteria to establish the ALS pathological staging. Using the source-reconstructed magnetoencephalography (MEG) approach, we demonstrated that global brain hyperconnectivity is associated with early and advanced clinical ALS stages. Using nuclear magnetic resonance (1H-NMR) and high resolution mass spectrometry (HRMS) spectroscopy, here we studied the metabolomic profile of ALS patients’ sera characterized by different stages of disease progression—namely early and advanced. Multivariate statistical analysis of the data integrated with the network analysis indicates that metabolites related to energy deficit, abnormal concentrations of neurotoxic metabolites and metabolites related to neurotransmitter production are pathognomonic of ALS in the advanced stage. Furthermore, analysis of the lipidomic profile indicates that advanced ALS patients report significant alteration of phosphocholine (PCs), lysophosphatidylcholine (LPCs), and sphingomyelin (SMs) metabolism, consistent with the exigency of lipid remodeling to repair advanced neuronal degeneration and inflammation.

SARS-CoV-2 is a virus belonging to the betacoronavirus family, causing fatal respiratory disease in humans, which became pandemic in 2020. Italy is one of the most affected countries by COVID-19, particularly in the northern regions. Several studies consider COVID-19 a zoonotic disease and, since Italy is the repository of a high biodiversity, SARS-CoV-2 infection in animals can be considered as a reservoir of the virus or favor the spreading between animals and humans. In this work, we analyzed the amino acid sequences of ACE2 protein of the most common domestic and wild animals present in Italy. Among the latter, we focused on ACE2 of the Chiroptera species present in Italy to identify the primary reservoir in this region. First, we reproduced in silico the Chiroptera ACE2/viral spike (S) protein interactions on the human ACE2/SARS-CoV-2 S complex model and identified the critical residues for the binding. In silico molecular docking of ACE2 belonging to Chiroptera vs SARS-CoV-2 S protein pointed to Rhinolophus ferrumequinum as a bat living in Italy, that may be a potential primary reservoir of the virus. On the other hand, a sequence similarity search on ACE2 of domestic and wild animals living in Italy pointed to domestic (horses, cats, cattle and sheep) and wild (European rabbits and grizzly bears) animal species as potential SARS-CoV-2 secondary reservoirs. Molecular docking of ACE2 belonging to these species vs S protein of Bat coronavirus (Bt-CoV/Rp3/2004) suggests that the primary reservoir Rhinolophus ferrumequinum may infect the secondary reservoirs, domestic and worldwide animals living in Italy, determining a specific risk of SARS-CoV-2 infection.

The supramolecular structure in peptides’ prolonged-released gel formulations is the most critical parameter for the determination of the pharmaceutical profile of the drug. Here, we report our investigation on lanreotide Autogel as a case study. For the first time, we describe the use of the pulsed field gradient (PFG) diffusion-ordered spectroscopy (DOSY) magic-angle spinning NMR to characterize the supramolecular self-assembly and molecular mobility of different samples of lanreotide Autogel formulations prepared according to different formulation protocols. The diffusion coefficient was used to calculate the hydrodynamic radii of supramolecular assemblies and build relative molecular models. DOSY data were integrated with NMR imaging (MRI) measurements and atomic force microscopy (AFM) imaging.

Alzheimer’s disease (AD) is a neurodegenerative pathology characterized by the presence of neurofibrillary tangles and amyloid plaques, the latter mainly composed of Aβ(1–40) and Aβ(1–42) peptides. The control of the Aβ aggregation process as a therapeutic strategy for AD has prompted the interest to investigate the conformation of the Aβ peptides, taking advantage of computational and experimental techniques. Mixtures composed of systematically different proportions of HFIP and water have been used to monitor, by NMR, the conformational transition of the Aβ(1–42) from soluble α-helical structure to β-sheet aggregates. In the previous studies, 50/50 HFIP/water proportion emerged as the solution condition where the first evident Aβ(1–42) conformational changes occur. In the hypothesis that this solvent reproduces the best condition to catch transitional helical-β-sheet Aβ(1–42) conformations, in this study, we report an extensive NMR conformational analysis of Aβ(1–42) in 50/50 HFIP/water v/v. Aβ(1–42) structure was solved by us, giving evidence that the evolution of Aβ(1–42) peptide from helical to the β-sheet may follow unexpected routes. Molecular dynamics simulations confirm that the structural model we calculated represents a starting condition for amyloid fibrils formation.

Feline immunodeficiency virus (FIV), a lentivirus causing an immunodeficiency syndrome in cats, represents a relevant model of pre-screening therapies for human immunodeficiency virus (HIV). The envelope glycoproteins gp36 in FIV and gp41 in HIV mediate the fusion of the virus with the host cell membrane. They have a common structural framework in the C-terminal region that includes a Trp-rich membrane-proximal external region (MPER) and a C-terminal heptad repeat (CHR). MPER is essential for the correct positioning of gp36 on the lipid membrane, whereas CHR is essential for the stabilization of the low-energy six-helical bundle (6HB) that is necessary for the fusion of the virus envelope with the cell membrane. Conformational data for gp36 are missing, and several aspects of the MPER structure of different lentiviruses are still debated. In the present work, we report the structural investigation of a gp36 construct that includes the MPER and part of the CHR domain (737-786gp36 CHR−MPER). Using 2D and 3D homo and heteronuclear NMR spectra on 15N and 13C double-labelled samples, we solved the NMR structure in micelles composed of dodecyl phosphocholine (DPC) and sodium dodecyl sulfate (SDS) 90/10 M: M. The structure of 737-786gp36 CHR−MPER is characterized by a helix−turn−helix motif, with a regular α-helix and a moderately flexible 310 helix, characterizing the CHR and the MPER domains, respectively. The two helices are linked by a flexible loop regulating their orientation at a ~43° angle. We investigated the positioning of 737-786gp36 CHR−MPER on the lipid membrane using spin label-enhanced NMR and ESR spectroscopies. On a different scale, using confocal microscopy imaging, we studied the effect of 737-786gp36 CHR−MPER on 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DOPC/DOPG) multilamellar vesicles (MLVs). This effect results in membrane budding and tubulation that is reminiscent of a membrane-plasticizing role that is typical of MPER domains during the event in which the virus envelope merges with the host cell membrane.

Feline immunodeficiency virus (FIV) is a naturally occurring Lentivirus causing acquired immunodeficiency syndrome in felines. It is considered a useful non-primate model to study HIV infection, and to test anti-HIV vaccine. Similarly to HIV, FIV enters cells via a mechanism involving a surface glycoprotein named gp36. C8 is a short synthetic peptide corresponding to the residues 770WEDWVGWI777 of gp36 membrane proximal external region (MPER). It elicits antiviral activity by inhibiting the fusion of the FIV and host cell membrane. C8 is endowed with evident membrane binding property, inducing alteration of the phospholipid bilayer and membrane fusion. The presence and the position of tryptophan residues in C8 are important for antiviral activity: the C8 derivative C6a, obtained by truncating the N-terminal 770WE771 residues, exhibits conserved antiviral activity, while the C8 derivative C6b, derived from the truncation of the C-terminal 776WI777, is nearly inactive. To elucidate the structural factors that induce the different activity profiles of C6a and C6b, in spite of their similarity, we investigated the structural behaviour of the two peptides in membrane mimicking environments. Conformational data on the short peptides C6a and C6b, matched to those of their parent peptide C8, allow describing a pharmacophore model of antiviral fusion inhibitors. This includes the essential structural motifs to design new simplified molecules overcoming the pharmacokinetic and high cost limitations affecting the antiviral entry inhibitors that currently are in therapy.