Bridging the past and the future of virology: Surface plasmon resonance as a powerful tool to investigate virus/host interactions.

Abstract Despite decades of antiviral drug research and development, viruses still remain a top global healthcare problem. Compared to eukaryotic cells, viruses are composed by a limited numbers of proteins that, nevertheless, set up multiple interactions with cellular components, allowing the virus to take control of the infected cell. Each virus/host interaction can be considered as a therapeutical target for new antiviral drugs but, unfortunately, the systematic study of a so huge number of interactions is time-consuming and expensive, calling for models overcoming these drawbacks. Surface plasmon resonance (SPR) is a label-free optical technique to study biomolecular interactions in real time by detecting reflected light from a prism-gold film interface. Launched 20 years ago, SPR has become a nearly irreplaceable technology for the study of biomolecular interactions. Accordingly, SPR is increasingly used in the field of virology, spanning from the study of biological interactions to the identification of putative antiviral drugs. From the literature available, SPR emerges as an ideal link between conventional biological experimentation and system biology studies functional to the identification of highly connected viral or host proteins that act as nodal points in virus life cycle and thus considerable as therapeutical targets for the development of innovative antiviral strategies

Cutting Edge: Proangiogenic Properties of Alternatively Activated Dendritic Cells

Abstract Angiogenesis plays an important role in tissue remodeling and repair during the late phase of inflammation. In the present study, we show that human dendritic cells (DC) that matured in the presence of anti-inflammatory molecules such as calcitriol, PGE2, or IL-10 (alternatively activated DC) selectively secrete the potent angiogenic cytokine vascular endothelial growth factor (VEGF) isoforms VEGF165 and VEGF121. No VEGF production was observed in immature or classically activated DC. Also, the capacity to produce VEGF was restricted to the myeloid DC subset. When implanted in the chick embryo chorioallantoic membrane, alternatively activated DC elicit a marked angiogenic response, which is inhibited by neutralizing anti-VEGF Abs and by the VEGFR-2 inhibitor SU5416. Therefore, alternatively activated DC may contribute to the resolution of the inflammatory reaction by promoting VEGF-induced angiogenesis

Fibroblast Growth Factor-2 Antagonist Activity and Angiostatic Capacity of Sulfated Escherichia coli K5 Polysaccharide Derivatives *

The angiogenic basic fibroblast growth factor (FGF2) interacts with tyrosine kinase receptors (FGFRs) and heparan sulfate proteoglycans (HSPGs) in endothelial cells. Here, we report the FGF2 antagonist and antiangiogenic activity of novel sulfated derivatives of the Escherichia coli K5 polysaccharide. K5 polysaccharide was chemically sulfated in N- and/or O-position after N-deacetylation. O-Sulfated and N,O-sulfated K5 derivatives with a low degree and a high degree of sulfation compete with heparin for binding to 125I-FGF2 with different potency. Accordingly, they abrogate the formation of the HSPG.FGF2.FGFR ternary complex, as evidenced by their capacity to prevent FGF2-mediated cell-cell attachment of FGFR1-overexpressing HSPG-deficient Chinese hamster ovary (CHO) cells to wild-type CHO cells. They also inhibited 125I-FGF2 binding to FGFR1-overexpressing HSPG-bearing CHO cells and adult bovine aortic endothelial cells. K5 derivatives also inhibited FGF2-mediated cell proliferation in endothelial GM 7373 cells and in human umbilical vein endothelial (HUVE) cells. In all these assays, the N-sulfated K5 derivative and unmodified K5 were poorly effective. Also, highly O-sulfated and N,O-sulfated K5 derivatives prevented the sprouting of FGF2-transfected endothelial FGF2-T-MAE cells in fibrin gel and spontaneous angiogenesis in vitro on Matrigel of FGF2-T-MAE and HUVE cells. Finally, the highly N,O-sulfated K5 derivative exerted a potent antiangiogenic activity on the chick embryo chorioallantoic membrane. These data demonstrate the possibility of generating FGF2 antagonists endowed with antiangiogenic activity by specific chemical sulfation of bacterial K5 polysaccharide. In particular, the highly N,O-sulfated K5 derivative may provide the basis for the design of novel angiostatic compounds

Surface functionalization of extracellular vesicle nanoparticles with antibodies: a first study on the protein corona "variable"

To be profitably exploited in medicine, nanosized systems must be endowed with biocompatibility, targeting capability, the ability to evade the immune system, and resistance to clearance. Currently, biogenic nanoparticles, such as extracellular vesicles (EVs), are intensively investigated as the platform that naturally recapitulates these highly needed characteristics. EV native targeting properties and pharmacokinetics can be further augmented by decorating the EV surface with specific target ligands as antibodies. However, to date, studies dealing with the functionalization of the EV surface with proteins have never considered the protein corona "variable", namely the fact that extrinsic proteins may spontaneously adsorb on the EV surface, contributing to determine the surface, and in turn the biological identity of the EV. In this work, we explore and compare the two edge cases of EVs modified with the antibody Cetuximab (CTX) by chemisorption of CTX (through covalent binding via biorthogonal click-chemistry) and by formation of a physisorbed CTX corona. The results indicate that (i) no differences exist between the two formulations in terms of binding affinity imparted by molecular recognition of CTX versus its natural binding partner (epidermal growth factor receptor, EGFR), but (ii) significant differences emerge at the cellular level, where CTX-EVs prepared by click chemistry display superior binding and uptake toward target cells, very likely due to the higher robustness of the CTX anchorage

Treatment with FRAX486 rescues neurobehavioral and metabolic alterations in a female mouse model of CDKL5 deficiency disorder

Introduction: CDKL5 deficiency disorder (CDD) is a rare neurodevelopmental condition, primarily affecting girls for which no cure currently exists. Neuronal morphogenesis and plasticity impairments as well as metabolic dysfunctions occur in CDD patients. The present study explored the potential therapeutic value for CDD of FRAX486, a brain-penetrant molecule that was reported to selectively inhibit group I p21-activated kinases (PAKs), serine/threonine kinases critically involved in the regulation of neuronal morphology and glucose homeostasis.Methods: The effects of treatment with FRAX486 on CDD-related alterations were assessed in vitro (100 nM for 48h) on primary hippocampal cultures from Cdkl5-knockout male mice (Cdkl5-KO) and in vivo (20 mg/Kg, s.c. for 5 days) on Cdkl5-KO heterozygous females (Cdkl5-Het).Results: The in vitro treatment with FRAX486 completely rescued the abnormal neuronal maturation and the number of PSD95-positive puncta in Cdkl5-KO mouse neurons. In vivo, FRAX486 normalized the general health status, the hyperactive profile and the fear learning defects of fully symptomatic Cdkl5-Het mice. Systemically, FRAX486 treatment normalized the levels of reactive oxidizing species in the whole blood and the fasting-induced hypoglycemia displayed by CdklS-Het mice. In the hippocampus of Cdkl5-Het mice, treatment with FRAX486 rescued spine maturation and PSD95 expression and restored the abnormal PAKs phosphorylation at sites which are critical for their activation (P-PAK-Ser144/141/139) or for the control cytoskeleton remodeling (P-PAK1-Thr212).Conclusions: Present results provide evidence that PAKs may represent innovative therapeutic targets for CDD

An innovative strategy to investigate microbial protein modifications in a reliable fast and sensitive way: A therapy oriented proof of concept based on UV-C irradiation of SARS-CoV-2 spike protein

: The characterization of modifications of microbial proteins is of primary importance to dissect pathogen lifecycle mechanisms and could be useful in identifying therapeutic targets. Attempts to solve this issue yielded only partial and non-exhaustive results. We developed a multidisciplinary approach by coupling in vitro infection assay, mass spectrometry (MS), protein 3D modelling, and surface plasma resonance (SPR). As a proof of concept, the effect of low UV-C (273 nm) irradiation on SARS-CoV-2 spike (S) protein was investigated. Following UV-C exposure, MS analysis identified, among other modifications, the disruption of a disulphide bond within the conserved S2 subunit of S protein. Computational analyses revealed that this bond breakage associates with an allosteric effect resulting in the generation of a closed conformation with a reduced ability to bind the ACE2 receptor. The UV-C-induced reduced affinity of S protein for ACE2 was further confirmed by SPR analyses and in vitro infection assays. This comprehensive approach pinpoints the S2 domain of S protein as a potential therapeutic target to prevent SARS-CoV-2 infection. Notably, this workflow could be used to screen a wide variety of microbial protein domains, resulting in a precise molecular fingerprint and providing new insights to adequately address future epidemics

Integrated care for older multimorbid heart failure patients:protocol for the ESCAPE randomized trial and cohort study

ESCAPE Evaluation of a patient-centred biopsychosocial blended collaborative care pathway for the treatment of multimorbid elderly patients. Therapeutic Area Healthcare interventions for the management of older patients with multiple morbidities. Aims Multi-morbidity treatment is an increasing challenge for healthcare systems in ageing societies. This comprehensive cohort study with embedded randomized controlled trial tests an integrated biopsychosocial care model for multimorbid elderly patients. Hypothesis A holistic, patient-centred pro-active 9-month intervention based on the blended collaborative care (BCC) approach and enhanced by information and communication technologies can improve health-related quality of life (HRQoL) and disease outcomes as compared with usual care at 9 months. Methods Across six European countries, ESCAPE is recruiting patients with heart failure, mental distress/disorder plus ≥2 medical co-morbidities into an observational cohort study. Within the cohort study, 300 patients will be included in a randomized controlled assessor-blinded two-arm parallel group interventional clinical trial (RCT). In the intervention, trained care managers (CMs) regularly support patients and informal carers in managing their multiple health problems. Supervised by a clinical specialist team, CMs remotely support patients in implementing the treatment plan—customized to the patients' individual needs and preferences—into their daily lives and liaise with patients' healthcare providers. An eHealth platform with an integrated patient registry guides the intervention and helps to empower patients and informal carers. HRQoL measured with the EQ-5D-5L as primary endpoint, and secondary outcomes, that is, medical and patient-reported outcomes, healthcare costs, cost-effectiveness, and informal carer burden, will be assessed at 9 and ≥18 months. Conclusions If proven effective, the ESCAPE BCC intervention can be implemented in routine care for older patients with multiple morbidities across the participating countries and beyond

Polyanionic Drugs and Viral Oncogenesis: a Novel Approach to Control Infection, Tumor-associated Inflammation and Angiogenesis

Polyanionic macromolecules are extremely abundant both in the extracellular environment and inside the cell, where they are readily accessible to many proteins for interactions that play a variety of biological roles. Among polyanions, heparin, heparan sulfate proteoglycans (HSPGs) and glycosphingolipids (GSLs) are widely distributed in biological fluids, at the cell membrane and inside the cell, where they are implicated in several physiological and/or pathological processes such as infectious diseases, angiogenesis and tumor growth. At a molecular level, these processes are mainly mediated by microbial proteins, cytokines and receptors that exert their functions by binding to HSPGs and/or GSLs, suggesting the possibility to use polyanionic antagonists as efficient drugs for the treatment of infectious diseases and cancer. Polysulfated (PS) or polysulfonated (PSN) compounds are a heterogeneous group of natural, semi-synthetic or synthetic molecules whose prototypes are heparin and suramin. Different structural features confer to PS/PSN compounds the capacity to bind and inhibit the biological activities of those same heparin-binding proteins implicated in infectious diseases and cancer. In this review we will discuss the state of the art and the possible future development of polyanionic drugs in the treatment of infectious diseases and cancer