Tolcapone, a clinically approved drug for the treatment of Parkinson's disease as an adjunct therapy, has recently emerged as a potential modulator of amyloid-β aggregation and toxicity, which are hallmark features of Alzheimer's disease and are also involved in ocular neurodegenerative disorders, including glaucoma and age-related macular degeneration. Despite these noteworthy findings, the molecular basis of the interaction between amyloid-β and tolcapone remains poorly understood, and the mechanisms by which tolcapone affects metal-amyloid-β species have yet to be explored. In this work, we investigate the binding interactions of tolcapone with both copper-free amyloid-β and copper-associated amyloid-β complexes, using a combination of techniques including UV-vis spectroscopy, circular dichroism, mass spectrometry, and surface plasmon resonance. The results reveal that tolcapone binds directly to amyloid-β monomers. Furthermore, in vitro assays confirm the capacity of tolcapone to act as a radical scavenger and to compete with amyloid-β for the binding of copper ions. Altogether, our findings suggest that tolcapone exerts a multifaceted protective effect, potentially inhibiting toxic metal-free and metal aggregation pathways by preventing metal coordination to amyloid-β or disrupting preformed amyloid-β-metal complexes, thus offering new perspectives to explore and develop its analogs for the treatment of neurodegenerative disorders.

Background: Elevated intraocular pressure in primary open-angle glaucoma (POAG) primarily stems from impaired aqueous humor (AH) outflow dynamics and aberrant extracellular matrix remodeling within the trabecular meshwork. Although uncovering global proteome perturbations is essential, conventional shotgun proteomics fails to resolve the structural states and site-specific proteolytic processing events driving these pathogenic alterations. Methods: Here, we present a site-resolved degradomic and proteomic characterization of AH from POAG patients and cataract control subjects at single-subject resolution ( ; 30 POAG, 36 controls), utilizing an integrated framework of data-independent acquisition (DIA) proteomics and TMTpro™ Zero-based N-terminomics coupled with orthogonal molecular biology validation. Findings: We identified a POAG-specific proteolytic fingerprint defined by the plasmin-dependent cleavage of the fibulin-1c isoform. Biochemical validation confirmed that disruption of this microfibrillar scaffolding axis destabilizes the large latent complex, triggering the pathological solubilization and accumulation of the Latency-Associated Peptide (LAP)–TGF-β precursor in glaucomatous AH. Interpretation: Our multi-dimensional dataset uncovers a finely tuned mechano-proteolytic axis underlying trabecular meshwork failure, establishing plasmin-mediated fibulin-1c processing and TGF-β complex destabilization as potential mechanistic targets for therapeutic intervention in POAG.

Long-lived proteins provide a uniquely informative substrate for studying the molecular chemistry of human ageing, and the eye offers one of the most accessible and spatially resolved systems for examining this process. The lens remains the principal model: its central crystallins are synthesized during embryonic and early postnatal life and are retained throughout the lifespan, thereby preserving a cumulative record of irreversible chemical damage. We then extend this framework to other ocular compartments that contain long-lived or slowly turned-over proteins, including Bruch’s membrane, the lens capsule and membrane proteome, the trabecular meshwork, and the corneal stroma, where cumulative chemical damage to structural proteins and extracellular matrices may likewise shape tissue ageing and disease susceptibility. With age, these proteins acquire extensive post-translational modifications, including deamidation, isoaspartate formation, racemization, truncation, oxidation, disulfide rearrangement, glycation, carbamylation, and photochemical adducts, which progressively reshape their conformational landscape.This review, we propose that the progressive, cumulative effect of these modifications constitutes a process defined as conformational drift: a chemically encoded displacement of protein ensembles away from their native conformational states toward heterogeneous, destabilized, poorly soluble, and aggregation-prone forms. For instance, in the lens, this process disrupts crystallin packing, depletes the chaperone reserve of α-crystallin, promotes phase separation and light scattering, and contributes to age-related stiffening of the lens nucleus. Finally, we critically evaluate current analytical strategies for mapping these molecular events and argue that the human eye offers an underused model for mechanistic geroscience beyond ophthalmology.

Background/Objectives: Citicoline, also known as CDP-choline, is a nootropic agent currently used in the treatment of glaucoma and is undergoing evaluation as a first-line therapy in a multi-center, international, phase III, randomized clinical trial involving citicoline eyedrops (ClinicalTrials.gov ID: NCT05710198). Numerous clinical and preclinical studies have linked the neuroenhancement and neuroprotective effects of citicoline to its role as a metabolic precursor for structural and functional components of cell membranes (such as phosphatidylcholine and sphingomyelin) and for neurotransmitters (e.g., acetylcholine and dopamine). However, compelling evidence suggests that the molecular mechanisms underlying its cytoprotective activity involve additional as-yet uncharacterized pharmacological actions. Methods: To further elucidate its pharmacology, we investigated the effect of two cytoprotective doses of citicoline (0.1 mM and 1 mM) on the global proteome of neuroblastoma cells using an unbiased shotgun proteomics approach. Results: With over 4000 unique proteins identified and quantified per experimental condition, the proteomics analysis revealed that citicoline, after 6 h of stimulation, induces a profound and robust remodeling of the intracellular proteome compared to untreated cells. Importantly, this effect was observed to significantly diminish by 18 h of stimulation, highlighting its transient nature (data are available via ProteomeXchange with identifier PXD061053). The clustering and rationalization of proteins upregulated by citicoline treatment identified the enrichment of key pathways for mRNA splicing, protein translation, proteostasis balance through the ubiquitin proteasome system (UPS), and mitochondrial metabolism. Conclusions: These proteomics findings introduce previously uncharacterized biological effects of citicoline and foster the working hypothesis that this drug may exert its cytoprotective activity through molecular mechanisms linked to the hormesis principle. These data further support the rationale for its clinical application in neurodegenerative processes and human disorders characterized by proteotoxicity.

Hexoses, particularly glucose, are one of the most essential molecules for sustaining life; therefore, reliable methods for their analysis are very important. In our study, we present a qualitative and quantitative approach for analysing hexoses using MALDI IMS (Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging) with betaine aldehyde derivatisation and a CHCA (α-Cyano-4-hydroxycinnamic acid) matrix in positive ionisation mode. In this study, we demonstrated betaine aldehyde derivatisation of glucose from dried droplets and explored the analysis of hexoses in brain and liver tissue slices. We assessed whether our method could distinguish between mannose, galactose, glucose, and fructose and optimised the preparation of a biomimetic calibration curve using stable-isotope labelled glucose for hexose analysis. For this purpose, we investigated the number of betaine aldehyde layers required to obtain a proper calibration curve; examined whether changes in the spray nozzle position during CHCA matrix deposition could facilitate analysis and investigated how storage conditions influenced the calibration curve analysis. Finally, we optimised the technique for liver and brain analysis and assessed variations in hexose levels between brain, liver, kidney, and spinal cord tissues from control and morphine-addicted animals. We hope that our biomimetic approach to creating the calibration curve will be helpful for quantitative analysis and aid in developing various quantitative methods for assessing endogenous substances.

Membraneless compartmentalization via liquid-liquid phase separation (LLPS) has emerged as a powerful strategy to organize biochemical reactions. Recently, peptide-based coacervates demonstrated the potential to function as microreactors by enhancing reaction kinetics through increased local concentrations and altered microenvironments. Here, we introduce an O-methylated diphenylalanine-based tripeptide LLLPFF-OCH3 containing an N-terminal proline, designed to undergo LLPS, and simultaneously function as an enantioselective organocatalyst. Comprehensive characterization via confocal microscopy, fluorescence recovery after photobleaching (FRAP), micro-Raman and attenuated total reflection infrared (ATR-IR) spectroscopy, diffusion-surface plasmon resonance (D-SPR), and molecular dynamics (MD) simulations revealed the formation of stable liquid droplets. In contrast, a racemic mixture of LLLPFF-OCH3 and DDDPFF-OCH3 failed to form liquid droplets and instead formed a solid precipitate, unveiling a critical role of enantiopurity in LLPS. Proof-of-concept catalytic studies proved enantioselective organocatalytic activity of the LLLPFF-OCH3 liquid coacervates. Beyond catalysis these results may have broader implications in understanding prebiotic chemistry and neurodegeneration.

Metal dyshomeostasis and oxidative stress are implicated in the progression of cancer, and neurodegenerative and peripheral aggregation‐related disorders. In this study, we investigated primaquine (PQ), a clinically used antimalarial drug, and PQ–lactobionic acid conjugate (LAPQ), a newly synthesized and characterized derivative obtained through conjugation with lactobionic acid, designed to improve physicochemical and biological properties. Both compounds were evaluated for their copper‐coordination ability, antioxidant properties, interaction with amyloid‐β (Aβ), and capacity to modulate reactive oxygen species (ROS)‐induced cellular damage. As a complementary biological line of investigation, their in vitro antiproliferative activity in the presence and absence of copper was also evaluated. The compounds directly interact with Aβ as demonstrated by surface plasmon resonance (SPR) studies. Biological studies revealed marked differences between the two molecules. PQ displayed intrinsic cytotoxicity, whereas LAPQ exhibited enhanced aqueous solubility and substantially reduced antiproliferative activity under the tested conditions, highlighting the impact of sugar conjugation. In cellular oxidative stress models, LAPQ showed a protective effect, preserving cell viability under ROS‐generating conditions. In summary, by interacting with Aβ and exhibiting antioxidant activity, two properties relevant to several neurodegenerative and peripheral aggregation‐related disorders, our novel compound LAPQ may provide a potential starting point for the development of therapies targeting Aβ–associated disorders.

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.

Ataxia-Telangiectasia (AT) is a rare disorder characterized by ATM deficiency and mitochondrial dysfunction. Because SIRT3 contributes to mitochondrial homeostasis in ATM-deficient settings, it represents a relevant molecular context for repurposing-oriented target-engagement studies. Here, we present an integrated computational and biophysical workflow designed to identify experimentally tractable SIRT3 binders within a clinically approved chemical space. A curated dataset of 2342 FDA-approved drugs was screened using hierarchical GNINA-based virtual screening, SAveRUNNER network analysis, triplicate 300 ns molecular dynamics simulations, post-MD redocking, MM/PBSA calculations, and experimental SPR validation. Four compounds were prioritized for further investigation: binimetinib, olaparib, mizolastine, and teniposide. Surface plasmon resonance (SPR) demonstrated direct binding of all four compounds to recombinant SIRT3, with equilibrium dissociation constants in the nano- to low-micromolar range. Binimetinib showed the highest apparent affinity (KD = 21.28 ± 3.5 nM), followed by olaparib (84 ± 5.7 nM), mizolastine (138 ± 3.5 nM), and teniposide (1470 ± 58 nM). Computational analyses supported the compatibility of these ligands with dynamically relaxed SIRT3 binding-site conformations and enabled the efficient prioritization of experimentally validated binders. These results support the value of an integrated in silico/SPR pipeline for identifying SIRT3-binding scaffolds. However, the functional consequences of binding, isoform selectivity, and biological relevance in ATM-deficient disease models remain to be established.

Ataxia-Telangiectasia (AT) is a rare disorder caused by ATM deficiency and characterized by genomic instability, mitochondrial dysfunction, and increased cancer susceptibility. Because SIRT3 contributes to mitochondrial homeostasis and metabolic adaptation in ATM-deficient settings, compounds that bind this target may provide useful starting points for repurposing-oriented therapeutic exploration. In this study, a curated dataset of 2,342 FDA-approved drugs was screened using an integrated workflow combining GNINA-based virtual screening, SAveRUNNER network analysis, 300 ns molecular dynamics simulations, post-MD redocking, MM/PBSA calculations, and experimental biophysical validation. Four compounds were prioritized for further investigation: binimetinib, olaparib, mizolastine, and teniposide. Surface plasmon resonance (SPR) demonstrated direct binding of all four compounds to recombinant SIRT3, with equilibrium dissociation constants in the nano- to submicromolar range. Binimetinib showed the highest apparent affinity (KD = 21.28 ±7 nM), followed by olaparib (84 ±8.1 nM), mizolastine (138 ±11 nM), and teniposide (811.25 ±5.3 nM). Computational analyses supported the compatibility of these ligands with dynamically relaxed SIRT3 binding-site conformations and enabled the efficient prioritization of experimentally tractable candidates. Overall, this study identifies four FDA-approved SIRT3-binding compounds and supports the value of an integrated computational/SPR workflow for repurposing-oriented hit identification in AT-related malignancy settings. Although these compounds displayed distinct translational profiles, the present results consistently indicated direct target engagement rather than definitive enzymatic inhibition, selectivity, or functional activity in disease-relevant models.

Graphene quantum dots (GQDs) exhibit size- and shape-dependent properties that critically influence their optical and electronic behavior, yet their reliable nanoscale characterization remains challenging. Here, we introduce a diffusion-based surface plasmon resonance (D-SPR) workflow that enables quantitative, shape-sensitive characterization of GQDs beyond conventional spherical approximations. Using sustainably synthesized GQDs derived from banana peels via optimized microwave-assisted methods, D-SPR resolves distinct particle populations, distinguishing monodisperse disk-like GQDs with average lateral dimensions of ≈2.5 nm from larger, polydisperse structures averaging ≈20 nm. These results are in excellent agreement with HR-TEM and DLS measurements. Crucially, unlike conventional DLS, D-SPR exploits diffusion-geometry coupling to directly identify non-spherical, disk-like GQD morphologies, supported by computational and mathematical modeling. This label-free approach delivers rapid, high-sensitivity size and shape resolution using minimal sample volumes, establishing D-SPR as a powerful complementary tool for the advanced characterization of carbon-based nanomaterials.

Glaucoma is a chronic optic neuropathy and is the second cause of irreversible blindness worldwide. Although the pathogenesis of the disease is not fully understood, the death of retinal ganglion cells and degeneration of the optic nerve are likely promoted by a combination of local and systemic factors. Growing attention has been paid to nonintraocular pressure risk factors, including mechanisms of inflammation and neuroinflammation. Phenotypical and molecular alterations of circulating immune cells, in particular, lymphocyte subsets, have been documented in murine models of glaucoma and in human subjects. Very recently, oxygen consumption rate and nicotinamide adenine dinucleotide levels of human peripheral blood mononuclear cells (PBMC) have been proposed as biomarkers of disease progression, thus suggesting that immune cells of glaucoma subjects present severe molecular and metabolic alterations. In this framework, this pilot study aimed to be the first to characterize global proteome perturbations of PBMC of patients with primary open-angle glaucoma (POAG) compared to nonglaucomatous controls (control) by shotgun proteomics. The approach identified >4,500 proteins and a total of 435 differentially expressed proteins between POAG and control subjects. Clustering and rationalization of proteomic data sets and immunodetection of selected proteins by Western blotting highlighted significant alterations of immune system compartments (i.e., complement factors, regulators of immune functions, and lymphocyte activation) and pathways serving key roles for immune system such as proteolysis (i.e., matrix metalloproteinases and their inhibitors), autophagy (i.e., beclin-1 and LC3B), cell proliferation (Bcl2), mitochondrial (i.e., sirtuin), and energetic/redox metabolism (i.e., NADK). Based on these findings, this proteomic study suggests that circulating immune cells suffer from heterogeneous alterations of central pathways involved in cell metabolism and homeostasis. Larger, properly designed studies are required to confirm specifically how immune cellular alterations may be involved in the pathogenesis of both neuroinflammation and glaucomatous disease.

Understanding the conformation and diffusion behavior of proteins in biological fluids is crucial for advancements in conformational disease research. While several experimental techniques are available for probing protein conformations, they often come with limitations, such as the need for fluorophores or specific experimental conditions. Albumin, one of the most abundant proteins in the blood and used as a tear supplement in treating ocular surface disorders, plays a vital role as a transport protein, binding various ligands and facilitating their transport. However, the direct relationship between albumin conformation and diffusion coefficient ( D ) in water solutions remains unexplored. In this study, we describe a novel Surface Plasmon Resonance technique coupled with Stochastic Gillespie′s algorithm simulations to correlate albumin D values with its conformational states directly. Our findings demonstrate the feasibility of monitoring albumin conformational changes under different environmental conditions, as well as its degradation kinetics by trypsin, by analyzing its diffusion characteristics, presenting a promising avenue for advancing our understanding of conformational diseases.

Analyzing diffusion processes within complex biomolecular mixtures is essential but technically challenging, especially for understanding molecular interactions in physiological environments. Here, we apply the diffusion-based surface plasmon resonance (D-SPR), a recent, label-free methodology that combines accurate SPR-based diffusion measurements with stochastic computational simulations, to address biomolecular complexity in ocular fluid models. By combining discrete Fréchet distance analysis with Gillespie algorithm simulations, D-SPR effectively detects unique diffusion patterns in binary (BSA/glycine, BSA/glucose) and ternary (HSA/ubiquitin/glycine) mixtures, enabling composition characterization without the need for external fluorophores or chromatographic techniques. Demonstrating the method sensitivity, as a proof-of-concept, we applied our workflow to probe the oligomeric transitions of bovine lens α-crystallin induced by mild acidification (pH 6.5). Our findings revealed subtle oligomer dissociation events and identified smaller, rapidly diffusing subunits, undetectable by conventional dynamic light scattering, thus providing insights into protein structural changes potentially pertinent to cataractogenesis. Considering the critical role of diffusion-based processes in eye health, the proposed D-SPR approach provides an advanced and versatile analytical tool with significant potential applications in medical research, especially ophthalmology.

The ubiquitin proteasome system is a critical regulator of proteostasis and shows altered activity and composition in neurodegenerative diseases affecting both the brain (e.g., Alzheimer's disease) and the retina/optic nerve (e.g., age-related macular degeneration, glaucoma). A common feature of neurodegeneration is the progressive accumulation of amyloidogenic proteins such as beta-amyloid and tau protein (MAPT gene). There is compelling evidence that the aggregation propensity of tau protein is regulated by post-synthetic modifications including phosphorylation and ubiquitylation. These alterations are gaining increasing pathological relevance not only for brain tauopathies but also for the retinal/optic nerve degenerative diseases. In this regard, site-specific mono-ubiquitylated (Ub) tau proteoforms, have been recently identified in neurodegenerative brains. In this work, the cleavage patterns of the uncapped 20S proteasome acting on mono-Ub regio-isomers of tauK18, which covers the 4RD domain, have been unveiled by using SpectraSage, a novel proteomics software conceived for the MS1 identification of complex branched peptide and here introduced for the first time. Ub position was found to affect regio-isomers susceptibility to proteolysis and unexpectedly long Ub-tauK18 branched peptides have been identified, proving distinct catalytic preferences. These findings show that the 20S digests mono-Ub proteins through specific enzymatic mechanisms and the implications of the latter on neurodegeneration are discussed.

Information regarding the dimension and the shape of molecules in solution represents a holy grail for chemists. Recently, newly designed surface plasmon resonance (SPR) methods to precisely measure the diffusion coefficients (D) (D-SPR) have been developed and applied successfully to a variety of molecules, ranging from diverse long-chain alcohols to protein conformers and oligomers involved in conformational disorders, mostly represented by neurodegenerative processes of nervous tissues of the brain and retina. The dependence of D on the molecular size, shape, and oligomerization state of different molecules has been widely investigated, opening up new avenues and tools for chemical, biochemical, and clinical research. Herein, the historical basis and the development of the newly proposed D-SPR method are briefly described to obtain meaningful information about molecular features that are otherwise hard to characterize using more common and traditional bioanalytical approaches.

Rhegmatogenous Retinal Detachment (RRD) is a severe ocular condition characterized by the detachment of the neurosensory retina from the retinal pigment epithelium and caused by retinal tears. Pars Plana Vitrectomy (PPV) is the standard surgical procedure for RRD, and is intended to remove the vitreous gel, which shapes the eye, and provides mechanical and nutritional support to the retina. The study of the vitreous proteome isolated from RRD patients may help decipher the pathobiology of the disease and that of its complications, such as proliferative vitreo-retinopathy (PVR), which predispose to recurrent retinal detachment (observed in 20% of cases), a sight threatening condition. Herein, we set up a pilot shot-gun proteomics study (Data are available via ProteomeXchange with identifier PXD057155) to investigate the perturbations of vitreous proteome comparing RRD patients (n = 8) to patients affected by idiopathic Epiretinal Membranes (ERM), used as controls (n = 8). Spectra were first searched and analyzed to identify proteome perturbations. Thereafter, starting from the hypothesis that RRD could be sustained by altered proteolytic processing of structural and non-structural elements of vitreous gel, N- and C-termini were mined to uncover endogenous proteolytic events. Our search revealed a comprehensive array of proteolytic events and sites across numerous proteins. While some of these were previously documented in different biological samples (like plasma, cell models, etc.), our findings point to new, potentially specific cleavage sites on macromolecular components unique to the vitreous fluid and retinal layers (e.g., interphotoreceptor matrix proteoglycan 1 and 2). Comparison between the N- and C-termini landscapes and the perturbations of global proteome highlighted robust alterations of the repertoire of cleaved proteins between RRD patients and reference control subjects. Additional immunoblotting studies on a selection of proteins envisage that RRD is characterized by unbalanced proteolysis of structural and non-structural components involved in the regulation of immune processes, proteolytic control and, particularly, angiogenesis.

Implementations of matrix multiplication via diffusion and reactions, thus eliminating the need for electronics, have been proposed as a stepping stone to realize molecular nano-neural networks (M3N). This work examines whether such "matrix multiplication units" can function spontaneously, i.e., without continuous external energy input. We employ the theory of local non-equilibrium thermodynamics in the linear regime, modeling the system through coupled reaction-diffusion equations and deriving the resulting entropy production. Numerical simulations on a 2D computational mesh confirm that correct matrix multiplication and strictly increasing entropy can be attained under two key conditions: negligible cross-diffusion among distinct species and sufficiently sharp membranes to prevent back diffusion. When these constraints are met, the system concentrations naturally converge to the desired results, suggesting that autonomous chemical computing can be realized if the design parameters align with thermodynamic requirements.

NH2 decorated intrinsically photoluminescent hydrogels (IPH-NH2) were functionalized with the addition of various peptides via EDC/NHS coupling method. These peptidic devices bind copper with binding affinities depending on surface functionalization. Particularly, fluorescence analysis of copper titrations, alongside the determination of quenching efficiency and lifetime measurements, allowed to assess binding constants and to elucidate the underlying binding mechanism. Various peptides, having the same copper binding amino acidic residues (GHK) but different chain lengths, were tested and it was found that increasing the distance of the GHK sequence from the IPH-NH2 surface resulted in a decrease in the binding constant, as well as a reduction in quenching efficiency, whereas the binding mechanism remained unchanged as indicated by lifetime measurements. This method not only provides binding constants for peptides immobilized on biosensor surfaces or pre-fabricated devices without altering their structure, but also contributes to the optimization of biosensor design, tailoring it to its intended application.