Label-free determination of diffusion coefficients at the nanoscale through modelling of the Surface Plasmon Resonance signal.

Surface plasmon resonance (SPR) is normally used to measure the kinetic parameters of biomolecular interactions between a molecule immobilized on a gold surface and another one flowing in a microfluidic channel above the surface. During the SPR measurements, convection-diffusion phenomena occur inside the microfluidic channels, but they are generally minimized by appropriate experimental setup in order to obtain diffusion free kinetic parameters of the molecular interactions. In this work, for the first time, a commercial SPR apparatus has been used to obtain non canonical scientific parameters.

The Delayed Turnover of Proteasome Processing of Myocilin upon Dexamethasone Stimulation Introduces the Profiling of Trabecular Meshwork Cells' Ubiquitylome.

Glaucoma is chronic optic neuropathy whose pathogenesis has been associated with the altered metabolism of Trabecular Meshwork Cells, which is a cell type involved in the synthesis and remodeling of the trabecular meshwork, the main drainage pathway of the aqueous humor. Starting from previous findings supporting altered ubiquitin signaling, in this study, we investigated the ubiquitin-mediated turnover of myocilin (MYOC/TIGR gene), which is a glycoprotein with a recognized role in glaucoma pathogenesis, in a human Trabecular Meshwork strain cultivated in vitro in the presence of dexamethasone. This is a validated experimental model of steroid-induced glaucoma, and myocilin upregulation by glucocorticoids is a phenotypic marker of Trabecular Meshwork strains.

Surface Plasmon Resonance Allows to Correlate Molecular Properties With Diffusion Coefficients of Linear Chain Alcohols.

Every biological and physicochemical process occurring in a fluid phase depends on the diffusion coefficient (D) of the species in solution. In the present work, a model to describe and fit the behaviour of as a function of structure and extensive thermodynamics parameters in binary solutions of linear chain organic molecules is developed. Supporting experimental and computational evidences for this model are obtained by measuring for a series of -alcohols through a novel surface plasmon resonance method and molecular dynamics simulations.

Carbon dots fluorescence can be used to distinguish isobaric peptide and to monitor protein oligomerization dynamics.

Carbon dots (CD) are widely investigated particles with interesting fluorescent properties which are reported to be used for various purposes, as they are biocompatible, resistant to photobleaching and with tuneable properties depending on the specific CD surface chemistry. In this work, we report on the possibility to use opportunely designed CD to distinguish among isobaric peptides almost undistinguishable by mass spectrometry, as well as to monitor protein aggregation phenomena. Particularly, cell-penetrating peptides containing the carnosine moiety at different positions in the peptide chain produce sequence specific fluorescent signals.

Detection of insulin oligomeric forms by a novel surface plasmon resonance-diffusion coefficient based approach.

Insulin is commonly used to treat diabetes and undergoes aggregation at the site of repeated injections in diabetic patients. Moreover, aggregation is also observed during its industrial production and transport and should be avoided to preserve its bioavailability to correctly adjust glucose levels in diabetic patients. However, monitoring the effect of various parameters (pH, protein concentration, metal ions, etc.

Fraisinib: a calixpyrrole derivative reducing A549 cell-derived NSCLC tumor acts as a ligand of the glycine-tRNA synthase, a new molecular target in oncology.

Lung cancer is the leading cause of death in both men and women, constituting a major public health problem worldwide. Non-small-cell lung cancer accounts for 85%-90% of all lung cancers. We propose a compound that successfully fights tumor growth by targeting the enzyme GARS1.

The Insulin-Degrading Enzyme from Structure to Allosteric Modulation: New Perspectives for Drug Design.

The insulin-degrading enzyme (IDE) is a Zn peptidase originally discovered as the main enzyme involved in the degradation of insulin and other amyloidogenic peptides, such as the β-amyloid (Aβ) peptide. Therefore, a role for the IDE in the cure of diabetes and Alzheimer's disease (AD) has been long envisaged. Anyway, its role in degrading amyloidogenic proteins remains not clearly defined and, more recently, novel non-proteolytic functions of the IDE have been proposed.

Mediterranean Diet, Sustainability, and Tourism-A Study of the Market's Demand and Knowledge.

Globalization intensified competitiveness among agribusinesses worldwide in recent years. The European Commission focused on enhancing sustainable agriculture and food products' territorial uniqueness for competing in the international market. The Mediterranean diet (MD) is a model of feeding and lifestyle belonging to the ancient Mediterranean culture, which also embodies a sustainable food system.

Time-of-flight SIMS investigation of peptides containing cell penetrating sequences.

Surface functionalization with biological molecules, such as peptides or proteins, is a very promising method for developing new biomaterials with many potential applications. However, due to their chemical complexity, the characterization of biological materials is often a very challenging task. In this context, time-of-flight secondary ion mass spectrometry is a very helpful characterization tool due to its ability to provide very detailed spatially resolved chemical information of the topmost layer.

Insights into the binding of Ag ions with SilE model peptides: an NMR and MS coupled approach.

The diffuse and renewed use of silver as antimicrobial agent has caused the development of resistance to silver ions in some bacterial strains, posing a serious threat for health systems. In order to cast light on the mechanistic features of resistance, here, we aimed to understand how silver interacts with the periplasmic metal-binding protein SilE which is engaged in bacterial silver detoxification. This aim was addressed by studying two peptide portions of SilE sequence (SP2 and SP3) that contain the putative motifs involved in Ag+ binding.

Carbon dots as a versatile tool to monitor insulin aggregation.

The possibility to monitor peptide and protein aggregation is of paramount importance in the so-called conformational diseases, as the understanding of many physiological pathways, as well as pathological processes involved in the development of such diseases, depends very much on the actual possibility to monitor biomolecule oligomeric distribution and aggregation. In this work, we report a novel experimental method to monitor protein aggregation, based on the change of the fluorescent properties of carbon dots upon protein binding. The results obtained in the case of insulin with this newly proposed experimental approach are compared with those obtained with other common experimental techniques normally used for the same purpose (circular dichroism, DLS, PICUP and ThT fluorescence).

GxxxG Motif Stabilize Ion-Channel like Pores through C-H···O Interaction in Aβ (1-40).

Aβ (1-40) can transfer from the aqueous phase to the bilayer and thus form stable ion-channel-like pores where the protein has alpha-helical conformation. The stability of the pores is due to the presence of the GXXXG motif. It has been reported that these ion-channel-like pores are stabilized by a Cα-H···O hydrogen bond that is established between a glycine of the GXXXG sequence of an alpha-helix and another amino acid of a vicinal alpha-helix.

The Use of Surface Plasmon Resonance to Study the Interactions of Proteins Involved in Conformational Diseases: Experimental Approaches for New Therapeutical Perspectives.

In recent years, the scientific community has been trying to tackle different diseases by using unifying and holistic approaches based on the concept that it is possible to target apparently very different diseases under a comprehensive general scheme. In other words, various different diseases have been grouped together under the label of "conformational diseases", because the triggering cause for each malady is the misfolding of a specific protein, whose dyshomeostasis and accumulation cause all the other downhill biomolecular events characteristic of each different disease. In a parallel manner, analytical techniques have developed to investigate protein misfolding and accumulation, so as to give a valid technical support to the investigation of conformational diseases.

Dipyridamole for tracking amyloidogenic proteins aggregation and enhancing polyubiquitination.

Dipyridamole is currently used as a medication that inhibits blood clot formation and it is also investigated in the context of neurodegenerative and other amyloid related diseases. Here, we propose this molecule as a new diagnostic tool to follow the aggregation properties of three different amyloidogenic proteins tested (insulin, amylin and amyloid β peptide 1-40). Results show that dipyridamole is sensitive to early stage amyloid formation undetected by thioflavin T, giving a different response for the aggregation of the three different proteins.

Carbon dots surface chemistry drives fluorescent properties: New tools to distinguish isobaric peptides.

The possibility to design rational carbon dots surface functionalization for specific analytical and bioanalytical applications is hindered by the lack of a full knowledge of the surface chemical features driving fluorescent properties. In this model study, we have synthesized four different peptides, three of which are isobaric and not distinguishable by common MSMS experiments. After having characterized the peptides conformations by CD analyses, we have covalently bonded all four peptides to carbon dots by using different experimental procedures, which produce different functional groups on the carbon dots surface.

An SPR-based method for Hill coefficient measurements: the case of insulin-degrading enzyme.

Insulin-degrading enzyme (IDE) is a highly conserved zinc metallopeptidase and is capable to catalytically cleave several substrates besides insulin, playing a pivotal role in several different biochemical pathways. Although its mechanism of action has been widely investigated, many conundrums still remain, hindering the possibility to rationally design specific modulators which could have important therapeutical applications in several diseases such as diabetes and Alzheimer's disease. In this scenario, we have developed a novel surface plasmon resonance (SPR) method which allows for directly measuring the enzyme cooperativity for the binding of insulin in the presence of different IDE activity modulators: carnosine, ATP, and EDTA.

Neuroprotective Effect of Carnosine Is Mediated by Insulin-Degrading Enzyme.

l-Carnosine is an endogenous dipeptide that has high potential for therapeutic purposes, being an antioxidant with metal chelating, anti-aggregating, anti-inflammatory, and neuroprotective properties. Despite its potential therapeutic values, the biomolecular mechanisms involved in neuroprotection are not fully understood. Here, we demonstrate, at chemical and biochemical levels, that insulin-degrading enzyme plays a pivotal role in carnosine neuroprotection.

Insulin-Degrading Enzyme Is a Non Proteasomal Target of Carfilzomib and Affects the 20S Proteasome Inhibition by the Drug.

Carfilzomib is a last generation proteasome inhibitor (PI) with proven clinical efficacy in the treatment of relapsed/refractory multiple myeloma. This drug is considered to be extremely specific in inhibiting the chymotrypsin-like activity of the 20S proteasome, encoded by the β5 subunit, overcoming some bortezomib limitations, the first PI approved for multiple myeloma therapy which is however burdened by a significant toxicity profile, due also to its off-target effects. Here, molecular approaches coupled with molecular docking studies have been used to unveil that the Insulin-Degrading Enzyme, a ubiquitous and highly conserved Zn peptidase, often found to associate with proteasome in cell-based models, is targeted by carfilzomib in vitro.

The interplay between lipid and Aβ amyloid homeostasis in Alzheimer's Disease: risk factors and therapeutic opportunities.

Alzheimer's Diseases (AD) is characterized by the accumulation of amyloid deposits of Aβ peptide in the brain. Besides genetic background, the presence of other diseases and an unhealthy lifestyle are known risk factors for AD development. Albeit accumulating clinical evidence suggests that an impaired lipid metabolism is related to Aβ deposition, mechanistic insights on the link between amyloid fibril formation/clearance and aberrant lipid interactions are still unavailable.

The insulin-degrading enzyme as a link between insulin and neuropeptides metabolism.

We have applied a recently developed HPLC-MS enzymatic assay to investigate the cryptic peptides generated by the action of the insulin-degrading enzyme (IDE) on some neuropeptides (NPs) involved in the development of tolerance and dependence to opioids. Particularly, the tested NPs are generated from the NPFF precursor (pro-NPFF (A)): NPFF (FLFQPQRF) and NPAF (AGEGLSSPFWSLAAPQRF). The results show that IDE is able to cleave NPFF and NPAF, generating specific cryptic peptides.

Histological and Histomorphometric Evaluation of New Bone Formation after Maxillary Sinus Augmentation with Two Different Osteoconductive Materials: A Randomized, Parallel, Double-Blind Clinical Trial.

This study aimed to investigate the histological features of deproteinized equine bone mineral (DEBM) and anorganic bovine bone (ABB) after human sinus augmentation with the lateral approach. Twenty-three sinus augmentations were performed in 16 patients (male: 10/female: 6) using DEBM or ABB in a randomized fashion. Healing took place over the next 6 months.

Defective proteasome biogenesis into skin fibroblasts isolated from Rett syndrome subjects with MeCP2 non-sense mutations.

Rett Syndrome (RTT) is a rare X-linked neurodevelopmental disorder which affects about 1: 10000 live births. In >95% of subjects RTT is caused by a mutation in Methyl-CpG binding protein-2 (MECP2) gene, which encodes for a transcription regulator with pleiotropic genetic/epigenetic activities. The molecular mechanisms underscoring the phenotypic alteration of RTT are largely unknown and this has impaired the development of therapeutic approaches to alleviate signs and symptoms during disease progression.

THE USE OF MASS SPECTROMETRY TO STUDY ZN-METALLOPROTEASE-SUBSTRATE INTERACTIONS.

Zinc metalloproteases (ZnMPs) participate in diverse biological reactions, encompassing the synthesis and degradation of all the major metabolites in living organisms. In particular, ZnMPs have been recognized to play a very important role in controlling the concentration level of several peptides and/or proteins whose homeostasis has to be finely regulated for the correct physiology of cells. Dyshomeostasis of aggregation-prone proteins causes pathological conditions and the development of several different diseases.

Pyrazolones Activate the Proteasome by Gating Mechanisms and Protect Neuronal Cells from β-Amyloid Toxicity.

Proteasome malfunction parallels abnormal amyloid accumulation in Alzheimer's Disease (AD). Here we scrutinize a small library of pyrazolones by assaying their ability to enhance proteasome activity and protect neuronal cells from amyloid toxicity. Tube tests evidenced that aminopyrine and nifenazone behave as 20S proteasome activators.