Electrostatics promotes molecular crowding and selects the aggregation pathway in fibril-forming protein solutions

The role of intermolecular interaction in fibril-forming protein solutions and its relation with molecular conformation are crucial aspects for the control and inhibition of amyloid structures. Here, we study the fibril formation and the protein-protein interactions for two proteins at acidic pH, lysozyme and α-chymotrypsinogen. By using light scattering experiments and the Kirkwood-Buff integral approach, we show how concentration fluctuations are damped even at moderate protein concentrations by the dominant long-ranged electrostatic repulsion, which determines an effective crowded environment. In denaturing conditions, electrostatic repulsion keeps the monomeric solution in a thermodynamically metastable state, which is escaped through kinetically populated conformational sub-states. This explains how electrostatics acts as a gatekeeper in selecting a specific aggregation pathway

Multiple myeloma-derived exosomes are enriched of amphiregulin (AREG) and activate the epidermal growth factor pathway in the bone microenvironment leading to osteoclastogenesis

Background: Multiple myeloma (MM) is a clonal plasma cell malignancy associated with osteolytic bone disease. Recently, the role of MM-derived exosomes in the osteoclastogenesis has been demonstrated although the underlying mechanism is still unknown. Since exosomes-derived epidermal growth factor receptor ligands (EGFR) are involved in tumor-associated osteolysis, we hypothesize that the EGFR ligand amphiregulin (AREG) can be delivered by MM-derived exosomes and participate in MM-induced osteoclastogenesis. Methods: Exosomes were isolated from the conditioned medium of MM1.S cell line and from bone marrow (BM) plasma samples of MM patients. The murine cell line RAW264.7 and primary human CD14 + cells were used as osteoclast (OC) sources. Results: We found that AREG was specifically enriched in exosomes from MM samples and that exosomes-derived AREG led to the activation of EGFR in pre-OC, as showed by the increase of mRNA expression of its downstream SNAIL in both RAW264.7 and CD14 + cells. The presence of neutralizing anti-AREG monoclonal antibody (mAb) reverted this effect. Consequently, we showed that the effect of MM-derived exosomes on osteoclast differentiation was inhibited by the pre-treatment of exosomes with anti-AREG mAb. In addition, we demonstrated the ability of MM-derived AREG-enriched exosomes to be internalized into human mesenchymal stromal cells (MSCs) blocking osteoblast (OB) differentiation, increasing MM cell adhesion and the release of the pro-osteoclastogenic cytokine interleukin-8 (IL8). Accordingly, anti-AREG mAb inhibited the release of IL8 by MSCs suggesting that both direct and indirect effects are responsible for AREG-enriched exosomes involvement on MM-induced osteoclastogenesis. Conclusions: In conclusion, our data indicate that AREG is packed into MM-derived exosomes and implicated in OC differentiation through an indirect mechanism mediated by OBs

CD90+ liver cancer cells modulate endothelial cell phenotype through the release of exosomes containing H19 lncRNA.

BACKGROUND: CD90+ liver cancer cells have been described as cancer stem-cell-like (CSC), displaying aggressive and metastatic phenotype. Using two different in vitro models, already described as CD90+ liver cancer stem cells, our aim was to study their interaction with endothelial cells mediated by the release of exosomes. METHODS: Exosomes were isolated and characterized from both liver CD90+ cells and hepatoma cell lines. Endothelial cells were treated with exosomes, as well as transfected with a plasmid containing the full length sequence of the long non-coding RNA (lncRNA) H19. Molecular and functional analyses were done to characterize the endothelial phenotype after treatments. RESULTS: Exosomes released by CD90+ cancer cells, but not by parental hepatoma cells, modulated endothelial cells, promoting angiogenic phenotype and cell-to-cell adhesion. LncRNA profiling revealed that CD90+ cells were enriched in lncRNA H19, and released this through exosomes. Experiments of gain and loss of function of H19 showed that this LncRNA plays an important role in the exosome-mediated phenotype of endothelial cells. CONCLUSIONS: Our data indicate a new exosome-mediated mechanism by which CSC-like CD90+ cells could influence their tumor microenvironment by promoting angiogenesis. Moreover, we suggest the lncRNA H19 as a putative therapeutic target in hepatocellular carcinoma

Embelin binds to human neuroserpin and impairs its polymerisation

Neuroserpin (NS) is a serpin inhibitor of tissue plasminogen activator (tPA) in the brain. The polymerisation of NS pathologic mutants is responsible for a genetic dementia known as familial encephalopathy with neuroserpin inclusion bodies (FENIB). So far, a pharmacological treatment of FENIB, i.e. an inhibitor of NS polymerisation, remains an unmet challenge. Here, we present a biophysical characterisation of the effects caused by embelin (EMB a small natural compound) on NS conformers and NS polymerisation. EMB destabilises all known NS conformers, specifically binding to NS molecules with a 1:1 NS:EMB molar ratio without unfolding the NS fold. In particular, NS polymers disaggregate in the presence of EMB, and their formation is prevented. The NS/EMB complex does not inhibit tPA proteolytic activity. Both effects are pharmacologically relevant: firstly by inhibiting the NS polymerisation associated to FENIB, and secondly by potentially antagonizing metastatic processes facilitated by NS activity in the brain

Preliminary results of citraves™ effects on low density lipoprotein cholesterol and waist circumference in healthy subjects after 12 weeks: A pilot open-label study

Appropriate monitoring and control of modifiable risk factors, such as the level of lowdensity lipoprotein cholesterol (LDL-C) and other types of dyslipidemia, have an important role in the prevention of cardiovascular diseases (CVD). Recently, various nutraceuticals with lipid-lowering effects have gained attention. In addition to the plant-derived bioactive compounds, recent studies suggested that plant cells are able to release small lipoproteic structures named extracellular vesicles (EVs). The interaction between EVs and mammalian cells could lead to beneficial effects through anti-inflammatory and antioxidant activities. The present study aimed to assess the safety of the new patented plant-based product citraVes™, containing extracellular vesicles (EVs) from Citrus limon (L.) Osbeck juice, and to investigate its ability to modulate different CV risk factors in healthy subjects. A cohort of 20 healthy volunteers was recruited in a prospective open-label study. All participants received the supplement in a spray-dried formulation at a stable dose of 1000 mg/day for 3 months. Anthropometric and hematobiochemical parameters were analyzed at the baseline and after the follow-up period of 1 and 3 months. We observed that the supplement has an effect on two key factors of cardiometabolic risk in healthy subjects. A significant change in waist circumference was found in women after 4 (85.4 [79.9, 91.0] cm, p < 0.005) and 12 (85.0 [80.0, 90.0] cm, p < 0.0005) weeks, when compared to the baseline value (87.6 [81.7, 93.6] cm). No difference was found in men (baseline: 100.3 [95.4, 105.2] cm; 4 weeks: 102.0 [95.7, 108.3] cm; 12 weeks: 100.0 [95.3, 104.7] cm). The level of LDL-C was significantly lower at 12 weeks versus 4 weeks (p = 0.0064). Our study evaluated, for the first time, the effects of a natural product containing plant-derived EVs on modifiable risk factors in healthy volunteers. The results support the use of EV extracts to manage cardiometabolic risk factors successfully

Pharmacological Properties and Biological Functions of the GPR17 Receptor, a Potential Target for Neuro-Regenerative Medicine

In 2006, cells heterologously expressing the "orphan" receptor GPR17 were shown to acquire responses to both uracil nucleotides and cysteinyl-leukotrienes, two families of signaling molecules accumulating in brain or heart as a result of hypoxic/traumatic injuries. In subsequent years, evidence of GPR17 key role in oligodendrogenesis and myelination has highlighted it as a "model receptor" for new therapies in demyelinating and neurodegenerative diseases. The apparently contrasting evidence in the literature about the role of GPR17 in promoting or inhibiting myelination can be due to its transient expression in the intermediate stages of differentiation, exerting a pro-differentiating function in early oligodendrocyte precursor cells (OPCs), and an inhibitory role in late stage maturing cells. Meanwhile, several papers extended the initial data on GPR17 pharmacology, highlighting a "promiscuous" behavior of this receptor; indeed, GPR17 is able to respond to other emergency signals like oxysterols or the pro-inflammatory cytokine SDF-1, underlying GPR17 ability to adapt its responses to changes of the surrounding extracellular milieu, including damage conditions. Here, we analyze the available literature on GPR17, in an attempt to summarize its emerging biological roles and pharmacological properties

Preliminary Studies for the Preparation of Casein-loaded Liposomes to Inhibit A\u3b21-40 Fibrillogenesis

\u3b1s1-Casein is a natural protein which constitutes the most prevalent form of casein in bovine milk. \u3b1s1-Casein is amphiphilic, almost unfolded, and consist of two highly hydrophobic zones separated by an hydrophilic region. Previous studies have demonstrated the ability of \u3b1s1-Casein to inhibit in vitro the nucleation phase of amyloid \u3b2-peptide (A\u3b2) fibrillogenesis by sequestering A\u3b2 species on its surface. One of the main hallmark for Alzheimer Disease (AD) is the extracellular deposition in brain tissues of proteinaceous plaques, rich of well-ordered A\u3b2 peptide amyloid aggregates. Although such an evidence, oligomeric intermediates in the fibrillogenesis have been discovered to be the most toxic species able to interfere with membranes and disturbing the cell functioning. In this context, a challenging therapeutic approach could target such early toxic oligomeric species. In order to exploit the inhibiting action of \u3b1s1-Casein as a possible AD treatment, it is crucial to define a controlled method to efficiently load, protect and deliver the protein to the brain. Liposomes are spherical phospholipids-based vesicles characterized by excellent biocompatibility and biodegradability, low toxicity, ability to incorporate and protect both hydrophilic and hydrophobic drugs as well as to cross the Blood Brain Barrier in order to access the Central Nervous System. Based on all these considerations, novel proteoliposomes composed by phospholipids, cholesterol and \u3b1s1-Casein were prepared and characterized. The proteoliposome preparation protocol was optimized in order to obtain the best results. Nanosystems were characterized by different biophysics techniques, such as light scattering, zeta-potential, laurdan fluorescence, chromatography and AFM imaging