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. Indeed, a specifically designed SPR experimental setup is described for carrying out measurements of the diffusion coefficient (D) of molecules in solutions. The high precision and reproducibility of the approach, as well as the wide applicability of the newly proposed SPR based method for the measurement of D of many different molecules and biomolecules, are here demonstrated and illustrated in detail.

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. This allows to propose a kind of combined analysis to explain the dependence of on various thermodynamic and structural parameters. The results suggest that for small linear systems in the range from 0 to 200 g mol⁻¹ and under the assumption that the diffusive activation energy is a linear function of mass, is strictly dependent on the molecular shape and on the relative strength of the solute‐solvent intermolecular forces represented by a parameter named R. The newly proposed approach can be utilized to characterize and monitor progressive changes in physicochemical properties for any investigated species upon increasing the dimension of the aggregate/molecule along a certain direction.

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. Western blotting and native-gel electrophoresis first uncovered that, in the presence of dexamethasone, myocilin turnover by proteasome particles was slower than in the absence of the drug. Thereafter, co-immunoprecipitation, RT-PCR and gene-silencing studies identified STUB1/CHIP as a candidate E3-ligase of myocilin. In this regard, dexamethasone treatment was found to downregulate STUB1/CHIP levels by likely promoting its proteasome-mediated turnover. Hence, to strengthen the working hypothesis about global alterations of ubiquitin-signaling, the first profiling of TMCs ubiquitylome, in the presence and absence of dexamethasone, was here undertaken by diGLY proteomics. Application of this workflow effectively highlighted a robust dysregulation of key pathways (e.g., phospholipid signaling, β-catenin, cell cycle regulation) in dexamethasone-treated Trabecular Meshwork Cells, providing an ubiquitin-centered perspective around the effect of glucocorticoids on metabolism and glaucoma pathogenesis.

Site-specific ubiquitylation dictates how the 20S proteasome generates unique branched peptides from Ub-tauK18 protein.

Redox unbalance, a molecular trait common to neurodegenerative conditions and para-physiological processes like aging, is a critical factor in disease development and in exacerbating progression. The mechanism by which redox imbalance perturbs cellular homeostasis is strongly linked to the activity and function of the ubiquitin–proteasome system (UPS). The UPS, along with autophagy, is the primary intracellular proteolytic system, regulating targeted proteolysis and removing damaged proteins. Consequently, the UPS serves also as the first line of defense for cellular recovery following exposure to redox stressors. Paradoxically, the composition and function of the UPS can also be negatively targeted by redox unbalance through a vicious cycle. The alterations in redox balance and UPS biological mechanisms are involved in the etiopathogenesis of chronic eye disorders. These disorders encompass a diverse repertoire of pathologies affecting the retinal layers (e.g., age-related macular degeneration, diabetic retinopathy) and the optic nerve (e.g., glaucoma). Nowadays, the comprehension of the interplay between proteostasis and oxidative redox status remains pivotal for identifying new therapeutic approaches. Encouragingly, a number of anti-oxidant compounds have been reported to modulate proteasome activity against redox insults in vitro and in vivo. Furthermore, these compounds provide cytoprotective roles in both in vitro and animal models of eye diseases. Therefore, this review highlights recent research on the interplay of the UPS with oxidative stress in physio-pathological conditions, focusing on the onset and progression of ocular diseases, thereby providing new insights into UPS-oxidative stress interaction.

Purpose: To explore whether the proteome of aqueous collected during primary repair of rhegmatogenous retinal detachment (RD), differs between patients who experience recurrency (Recurrent RD Group) and those who do not (No Recurrent RD Group). Methods: The aqueous proteome collected during primary surgery of 13 patients undergoing Recurrent RD was compared with 11 age- and sex-matched patients successfully operated for rhegmatogenous RD with no recurrency after 12-month follow-up, regardless of surgical technique. A label-free shotgun proteomics approach identified and quantified the repertoire of aqueous proteins. Differential protein expression between groups was determined using the Limma moderated Bayesian t -test, followed by false discovery rate (FDR) validation using Storey q-test. Results: Aqueous profiling identified >800 unique proteins; 45 exclusive to the Recurrent RD group, 10 exclusive to the no Recurrent RD group, and 33 differently expressed between groups (log2fold-change≥∣0.57∣, FDR ≤ 0.05). Proteins upregulated in Recurrent RD patients, clearly pointed to mechanisms of cell:cell and cell:matrix adhesiveness and mechano-transduction signaling pathways. Upregulated proteins included extracellular matrix components such as type I and IV collagens, biglycan, proteoglycans, and cell-membrane adhesion molecules. Conclusion: The baseline aqueous composition of RD patients that will eventually develop recurrency, differs significantly from those who will not, and already contains molecular signatures that may help identify the risk of recurrency at the time of primary repair. While acknowledging the pilot nature of the study, findings of this study strongly suggest that Recurrent RD is associated with cell adhesiveness pathways early alterations, offering targets for prognostic assessment and therapy.