Plasma Lipidomics and Coronary Plaque Changes: A Substudy of the SMARTool Clinical Trial.

AIMS: To date, no studies have investigated the association between lipid species and coronary plaque changes over time, quantitatively assessed by serial imaging. We aimed to prospectively determine the association between lipid species quantified by plasma lipidomic analysis, with coronary plaque changes according to composition assessed by quantitative serial analysis of coronary computed tomography angiography (CTA). METHODS AND RESULTS: Patients with suspected coronary artery disease (CAD) undergoing baseline coronary CTA were prospectively enrolled by 7 EU Centers in the SMARTool study and submitted to clinical, molecular and coronary CTA re-evaluation at follow-up (interscan period 6.39 ± 1.17 years). From the 202 patients that were analysed in the SMARTool main clinical study, lipidomic analysis was performed in 154 patients before the baseline coronary CTA, and this group was included in the present study. Quantitative CTA analysis was performed by a separate core laboratory blinded from clinical data. In univariable analysis, no lipid species were significantly associated with annual total and calcified plaque changes. After adjusting for clinical variables at baseline and statin use, 3 lipid species were significantly associated with non-calcified plaque progression. In detail, cholesteryl ester (CE)(20:3), sphingomyelin (SM)(40:3) and SM(41:1) were found positively related to non-calcified plaque progression (Bonferroni adjusted P-value = 0.005, 0.016 and 0.004, respectively). CONCLUSION: The current study showed an independent relationship between specific lipid species determined by plasma lipidomic analysis, and non-calcified coronary plaque progression assessed by serial, quantitative coronary CTA analysis.

The Clinical Utility of the Saliva Proteome in Rare Diseases: A Pilot Study for Biomarker Discovery in Primary Sclerosing Cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a rare chronic inflammatory liver disease characterized by biliary strictures and cholestasis. Due to the lack of effective serological indicators for diagnosis and prognosis, in the present study, we examined the potentiality of the saliva proteome to comprehensively screen for novel biomarkers. METHODS: Saliva samples of PSC patients and healthy controls were processed and subsequently analyzed using a liquid chromatography-tandem mass spectrometry technique. A bioinformatic approach was applied to detect the differentially expressed proteins, their related biological functions and pathways, and the correlation with the clinical evidence in order to identify a possible marker for the PSC group. RESULTS: We identified 25 differentially expressed proteins in PSC patients when compared to the healthy control group. Among them, eight proteins exhibited area under the curve values up to 0.800, suggesting these saliva proteins as good discriminators between the two groups. Multiple positive correlations were also identified between the dysregulated salivary proteins and increased serum alkaline phosphatase levels and the presence of ulcerative colitis. Pathway analysis revealed significant enrichments in the immune system, neutrophil degranulation, and in the interleukine-17 signaling pathway. CONCLUSION: We demonstrated the potentiality of saliva as a useful biofluid to obtain a fingerprint of the pathology, suggesting disulfide-isomerase A3 and peroxiredoxin-5 as the better discriminating proteins in PSC patients. Hence, analysis of saliva proteins could become, in future, a useful tool in the screening of patients with suspected PSC.

Aptamer-based gold nanoparticle aggregates for ultrasensitive amplification-free detection of PSMA.

Early diagnosis is one of the most important factors in determining the prognosis in cancer. Sensitive detection and quantification of tumour-specific biomarkers have the potential to improve significantly our diagnostic capability. Here, we introduce a triggerable aptamer-based nanostructure based on an oligonucleotide/gold nanoparticle architecture that selectively disassembles in the presence of the biomarker of interest; its optimization is based also on in-silico determination of the aptamer nucleotides interactions with the protein of interest. We demonstrate this scheme for the case of Prostate Specific Membrane Antigen (PSMA) and PSMA derived from PSMA-positive exosomes. We tested the disassembly of the system by diameter and count rate measurements in dynamic light scattering, and by inspection of its plasmon resonance shift, upon addition of PSMA, finding appreciable differences down to the sub-picomolar range; this points towards the possibility that this approach may lead to sensors competitive with diagnostic biochemical assays that require enzymatic amplification. More generally, this scheme has the potential to be applied to a broad range of pathologies with specific identified biomarkers.

Fluorescence Lifetime Nanoscopy of Liposomal Irinotecan Onivyde: From Manufacturing to Intracellular Processing.

Onivyde was approved by the Food and Drug Administration (FDA) in 2015 for the treatment of solid tumors, including metastatic pancreatic cancer. It is designed to encapsulate irinotecan at high concentration, increase its blood-circulation lifetime, and deliver it to cells where it is enzymatically converted into SN-38, a metabolite with 100- to 1000-fold higher anticancer activity. Despite a rewarding clinical path, little is known about the physical state of encapsulated irinotecan within Onivyde and how this synthetic identity changes throughout the process from manufacturing to intracellular processing. Herein, we exploit irinotecan intrinsic fluorescence and fluorescence lifetime imaging microscopy (FLIM) to selectively probe the supramolecular organization of the drug. FLIM analysis on the manufacturer's formulation reveals the presence of two coexisting physical states within Onivyde liposomes: (i) gelated/precipitated irinotecan and (ii) liposome-membrane-associated irinotecan, the presence of which is not inferable from the manufacturer's indications. FLIM in combination with high-performance liquid chromatography (HPLC) and a membrane-impermeable dynamic quencher of irinotecan reveals rapid (within minutes) and complete chemical dissolution of the gelated/precipitated phase upon Onivyde dilution in standard cell-culturing medium with extensive leakage of the prodrug from liposomes. Indeed, confocal imaging and cell-proliferation assays show that encapsulated and nonencapsulated irinotecan formulations are similar in terms of cell-uptake mechanism and cell-division inhibition. Finally, 2-channel FLIM analysis discriminates the signature of irinotecan from that of its red-shifted SN-38 metabolite, demonstrating the appearance of the latter as a result of Onivyde intracellular processing. The findings presented in this study offer fresh insights into the synthetic identity of Onivyde and its transformation from production to in vitro administration. Moreover, these results serve as another validation of the effectiveness of FLIM analysis in elucidating the supramolecular organization of encapsulated fluorescent drugs. This research underscores the importance of leveraging advanced imaging techniques to deepen our understanding of drug formulations and optimize their performance in delivery applications.

Visceral adipose tissue and residual cardiovascular risk: a pathological link and new therapeutic options.

Obesity is a heterogeneous disease that affects almost one-third of the global population. A clear association has been established between obesity and cardiovascular disease (CVD). However, CVD risk is known to be related more to the local distribution of fat than to total body fat. Visceral adipose tissue (VAT) in particular has a high impact on CVD risk. This manuscript reviews the role of VAT in residual CV risk and the available therapeutic strategies for decreasing residual CV risk related to VAT accumulation. Among the many pathways involved in residual CV risk, obesity and particularly VAT accumulation play a major role by generating low-grade systemic inflammation, which in turn has a high prognostic impact on all-cause mortality and myocardial infarction. In recent years, many therapeutic approaches have been developed to reduce body weight. Orlistat was shown to reduce both weight and VAT but has low tolerability and many drug-drug interactions. Naltrexone-bupropion combination lowers body weight but has frequent side effects and is contraindicated in patients with uncontrolled hypertension. Liraglutide and semaglutide, glucagon-like peptide 1 (GLP-1) agonists, are the latest drugs approved for the treatment of obesity, and both have been shown to induce significant body weight loss. Liraglutide, semaglutide and other GLP-1 agonists also showed a positive effect on CV outcomes in diabetic patients. In addition, liraglutide showed to specifically reduce VAT and inflammatory biomarkers in obese patients without diabetes. GLP-1 agonists are promising compounds to limit inflammation in human visceral adipocytes.

Proteomic Modulation in TGF-ß-Treated Cholangiocytes Induced by Curcumin Nanoparticles.

Curcumin is a natural polyphenol that exhibits a variety of beneficial effects on health, including anti-inflammatory, antioxidant, and hepato-protective properties. Due to its poor water solubility and membrane permeability, in the present study, we prepared and characterized a water-stable, freely dispersible nanoformulation of curcumin. Although the potential of curcumin nanoformulations in the hepatic field has been studied, there are no investigations on their effect in fibrotic pathological conditions involving cholangiocytes. Exploiting an in vitro model of transforming growth factor-ß (TGF-ß)-stimulated cholangiocytes, we applied the Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS)-based quantitative proteomic approaches to study the proteome modulation induced by curcumin nanoformulation. Our results confirmed the well-documented anti-inflammatory properties of this nutraceutic, highlighting the induction of programmed cell death as a mechanism to counteract the cellular damages induced by TGF-ß. Moreover, curcumin nanoformulation positively influenced the expression of several proteins involved in TGF-ß-mediated fibrosis. Given the crucial importance of deregulated cholangiocyte functions during cholangiopathies, our results provide the basis for a better understanding of the mechanisms associated with this pathology and could represent a rationale for the development of more targeted therapies.

Understanding the role of coronary artery revascularization in patients with left ventricular dysfunction and multivessel disease.

Coronary artery disease (CAD) is the most common cause of heart failure with reduced ejection fraction (HFrEF). Advances and innovations in medical therapy have been shown to play a crucial role in improving the prognosis of patients with CAD and HFrEF; however, mortality rate in these patients remains high, and the role of surgical and/or percutaneous revascularization strategy is still debated. The Surgical Treatment for Ischemic Heart Failure (STICH) trial and the Revascularization for Ischemic Ventricular Dysfunction (REVIVED) trial have attempted to provide an answer to this issue. Nevertheless, the results of these two trials have generated further uncertainties. Their findings do not provide a definitive answer about the ideal clinical phenotype for surgical or percutaneous coronary revascularization and dispute the historical dogma on myocardial viability and the theory of myocardial hibernation, raising new questions about the proper selection of patients who are candidates for coronary revascularization. The aim of this review is to provide an overview on the actual available evidence of coronary artery revascularization in patients with CAD and left ventricular dysfunction and to suggest new insights on the proper selection and management strategies in this high-risk clinical setting.

Apolipoprotein E ε4 triggers neurotoxicity via cholesterol accumulation, acetylcholine dyshomeostasis, and PKCε mislocalization in cholinergic neuronal cells.

The Apolipoprotein E (ApoE) has been known to regulate cholesterol and ß-amyloid (Aß) production, redistribution, and elimination, in the central nervous system (CNS). The ApoE ε4 polymorphic variant leads to impaired brain cholesterol homeostasis and amyloidogenic pathway, thus representing the major risk factor for Alzheimer's Disease (AD). Currently, less is known about the molecular mechanisms connecting ApoE ε4-related cholesterol metabolism and cholinergic system degeneration, one of the main AD pathological features. Herein, in vitro cholinergic neuron models were developed in order to study ApoE neuronal expression and investigate the possible interplay between cholesterol metabolism and cholinergic pathway impairment prompted by ε4 isoform. Particularly, alterations specifically occurring in ApoE ε4-carrying neurons (i.e. increased intracellular ApoE, amyloid precursor protein (APP) and Aß levels, elevated apoptosis, and reduced cell survival) were recapitulated. ApoE ε4 expression was found to increase intracellular cholesterol accumulation, by regulating the related gene expression, while reducing cholesterol precursor acetyl-CoA, which in turn fuels the acetylcholine (ACh) synthesis route. In parallel, although the ACh intracellular signalling was activated, as demonstrated by the boosted extracellular ACh as well as increased IP3 and Ca2+, the PKCε activation via membrane translocation was surprisingly suppressed, probably explained by the cholesterol overload in ApoE ε4 neuron-like cells. Consequently, the PKC-dependent anti-apoptotic and neuroprotective roles results impaired, reliably adding to other causes of cell death prompted by ApoE ε4. Overall, the obtained data open the way to further critical considerations of ApoE ε4-dependent cholesterol metabolism dysregulation in the alteration of cholinergic pathway, neurotoxicity, and neuronal death.

Sodium-Glucose Cotransporter 2 Inhibitors in Patients with Diabetes and Coronary Artery Disease: Translating the Benefits of the Molecular Mechanisms of Gliflozins into Clinical Practice.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) were initially developed for the treatment of diabetes due to their antihyperglycemic activity. However, in the light of the most recent clinical studies, they are revolutionizing the approach to cardiovascular disease in patients with and without diabetes. We aimed to generate real-world data about the use of SGLT2i in patients with T2DM and coronary artery disease (CAD), focusing on their effectiveness in glycemic control, adherence, long-term efficacy, and safety outcomes. On the basis of the inclusion and exclusion criteria, 143 patients were enrolled. Patients were treated with canagliflozin (n = 33 patients; 23%), dapagliflozin (n = 52 patients, 36.4%), empagliflozin (n = 48 patients; 33.6%), or ertugliflozin (n = 10 patients; 7%) as monotherapy or in combination with other antidiabetic drugs. All patients performed a clinical visit, and their medical history, blood sampling, and anthropometric parameters were measured at discharge and at 1-year follow-up. The reduction in HbA1c % value at 12 months was significant (8.2 vs. 7.4; p < 0.001). Trends in body weight and body mass index also confirmed the positive effect of the treatment (p < 0.0001), as did the reduction in abdominal adiposity (expressed via waist circumference). At 1-year follow-up, 74.1% of patients were adherent to the treatment, and 81.1% were persistent to the treatment. A total of 27 patients (18.8%) had to discontinue treatment early due to drug intolerance caused by genitourinary infections (11.9%), the drub being permanently ineffective (HbA1c not at target or decreasing: 4.9%), or because of expressing. a desire not to continue (2%). No major drug-related adverse events (diabetic ketoacidosis, Fournier's gangrene, lower-limb amputations) occurred at follow-up, while MACE events occurred in 14 patients (9.8%). In real-world patients with T2DM and CAD, SGLT2i have been effective in long-term glycemic control and the improvement in anthropometric indices with good tolerance, high adherence, persistence to treatment, and no major adverse events at 1-year follow-up.

Response of a Human Lens Epithelial Cell Line to Hyperglycemic and Oxidative Stress: The Role of Aldose Reductase.

A common feature of different types of diabetes is the high blood glucose levels, which are known to induce a series of metabolic alterations, leading to damaging events in different tissues. Among these alterations, both increased polyol pathway flux and oxidative stress are considered to play relevant roles in the response of different cells. In this work, the effect on a human lens epithelial cell line of stress conditions, consisting of exposure to either high glucose levels or to the lipid peroxidation product 4-hydroxy-2-nonenal, is reported. The occurrence of osmotic imbalance, alterations of glutathione levels, and expression of inflammatory markers was monitored. A common feature of the two stress conditions was the expression of COX-2, which, only in the case of hyperglycemic stress, occurred through NF-κB activation. In our cell model, aldose reductase activity, which is confirmed as the only activity responsible for the osmotic imbalance occurring in hyperglycemic conditions, seemed to have no role in controlling the onset of the inflammatory phenomena. However, it played a relevant role in cellular detoxification against lipid peroxidation products. These results, in confirming the multifactorial nature of the inflammatory phenomena, highlight the dual role of aldose reductase as having both damaging but also protecting activity, depending on stress conditions.

Increasing cell culture density during a developmental window prevents fated rod precursors derailment toward hybrid rod-glia cells.

In proliferating multipotent retinal progenitors, transcription factors dynamics set the fate of postmitotic daughter cells, but postmitotic cell fate plasticity driven by extrinsic factors remains controversial. Transcriptome analysis reveals the concurrent expression by postmitotic rod precursors of genes critical for the Müller glia cell fate, which are rarely generated from terminally-dividing progenitors as a pair with rod precursors. By combining gene expression and functional characterisation in single cultured rod precursors, we identified a time-restricted window where increasing cell culture density switches off the expression of genes critical for Müller glial cells. Intriguingly, rod precursors in low cell culture density maintain the expression of genes of rod and glial cell fate and develop a mixed rod/Muller glial cells electrophysiological fingerprint, revealing rods derailment toward a hybrid rod-glial phenotype. The notion of cell culture density as an extrinsic factor critical for preventing rod-fated cells diversion toward a hybrid cell state may explain the occurrence of hybrid rod/MG cells in the adult retina and provide a strategy to improve engraftment yield in regenerative approaches to retinal degenerative disease by stabilising the fate of grafted rod precursors.

Linoleic acid potentiates CD8+ T cell metabolic fitness and antitumor immunity.

The metabolic state represents a major hurdle for an effective adoptive T cell therapy (ACT). Indeed, specific lipids can harm CD8+ T cell (CTL) mitochondrial integrity, leading to defective antitumor responses. However, the extent to which lipids can affect the CTL functions and fate remains unexplored. Here, we show that linoleic acid (LA) is a major positive regulator of CTL activity by improving metabolic fitness, preventing exhaustion, and stimulating a memory-like phenotype with superior effector functions. We report that LA treatment enhances the formation of ER-mitochondria contacts (MERC), which in turn promotes calcium (Ca2+) signaling, mitochondrial energetics, and CTL effector functions. As a direct consequence, the antitumor potency of LA-instructed CD8 T cells is superior in vitro and in vivo. We thus propose LA treatment as an ACT potentiator in tumor therapy.

Ribose Intake as Food Integrator: Is It a Really Convenient Practice?

Reports concerning the beneficial effects of D-ribose administration in cardiovascular and muscle stressful conditions has led to suggestions for the use of ribose as an energizing food supplement for healthy people. However, this practice still presents too many critical issues, suggesting that caution is needed. In fact, there are many possible negative effects of this sugar that we believe are underestimated, if not neglected, by the literature supporting the presentation of the product to the market. Here, the risks deriving from the use of free ribose as ATP source, forcing ribose-5-phosphate to enter into the pentose phosphate pathway, is emphasized. On the basis of the remarkable glycation capacity of ribose, the easily predictable cytotoxic effect of the molecule is also highlighted.

Molecular Characterization of Plasma HDL, LDL, and VLDL Lipids Cargos from Atherosclerotic Patients with Advanced Carotid Lesions: A Preliminary Report.

Carotid atherosclerosis represents a relevant healthcare problem, since unstable plaques are responsible for approximately 15% of neurologic events, namely transient ischemic attack and stroke. Although statins treatment has proven effective in reducing LDL-cholesterol and the onset of acute clinical events, a residual risk may persist suggesting the need for the detection of reliable molecular markers useful for the identification of patients at higher risk regardless of optimal medical therapy. In this regard, several lines of evidence show a relationship among specific biologically active plasma lipids, atherosclerosis, and acute clinical events. We performed a Selected Reaction Monitoring-based High Performance Liquid Chromatography-tandem Mass Spectrometry (SRM-based HPLC-MS/MS) analysis on plasma HDL, LDL, and VLDL fractions purified, by isopycnic salt gradient ultracentrifugation, from twenty-eight patients undergoing carotid endarterectomy, having either a "hard" or a "soft" plaque, with the aim of characterizing the specific lipidomic patterns associated with features of carotid plaque instability. One hundred and thirty lipid species encompassing different lipid (sub)classes were monitored. Supervised multivariate analysis showed that lipids belonging to phosphatidylethanolamine (PE), sphingomyelin (SM), and diacylglycerol (DG) classes mostly contribute to discrimination within each lipoprotein fraction according to the plaque typology. Differential analysis evidenced a significant dysregulation of LDL PE (38:6), SM (32:1), and SM (32:2) between the two groups of patients (adj. p-value threshold = 0.05 and log2FC ≥ |0.58|). Using this approach, some LDL-associated markers of plaque vulnerability have been identified, in line with the current knowledge of the key roles of these phospholipids in lipoprotein metabolism and cardiovascular disease. This proof-of-concept study reports promising results, showing that lipoprotein lipidomics may present a valuable approach for identifying new biomarkers of potential clinical relevance.

Metabolic-imaging of human glioblastoma live tumors: A new precision-medicine approach to predict tumor treatment response early.

Background: Glioblastoma (GB) is the most severe form of brain cancer, with a 12-15 month median survival. Surgical resection, temozolomide (TMZ) treatment, and radiotherapy remain the primary therapeutic options for GB, and no new therapies have been introduced in recent years. This therapeutic standstill is primarily due to preclinical approaches that do not fully respect the complexity of GB cell biology and fail to test efficiently anti-cancer treatments. Therefore, better treatment screening approaches are needed. In this study, we have developed a novel functional precision medicine approach to test the response to anticancer treatments in organoids derived from the resected tumors of glioblastoma patients. Methods: GB organoids were grown for a short period of time to prevent any genetic and morphological evolution and divergence from the tumor of origin. We chose metabolic imaging by NAD(P)H fluorescence lifetime imaging microscopy (FLIM) to predict early and non-invasively ex-vivo anti-cancer treatment responses of GB organoids. TMZ was used as the benchmark drug to validate the approach. Whole-transcriptome and whole-exome analyses were performed to characterize tumor cases stratification. Results: Our functional precision medicine approach was completed within one week after surgery and two groups of TMZ Responder and Non-Responder tumors were identified. FLIM-based metabolic tumor stratification was well reflected at the molecular level, confirming the validity of our approach, highlighting also new target genes associated with TMZ treatment and identifying a new 17-gene molecular signature associated with survival. The number of MGMT gene promoter methylated tumors was higher in the responsive group, as expected, however, some non-methylated tumor cases turned out to be nevertheless responsive to TMZ, suggesting that our procedure could be synergistic with the classical MGMT methylation biomarker. Conclusions: For the first time, FLIM-based metabolic imaging was used on live glioblastoma organoids. Unlike other approaches, ex-vivo patient-tailored drug response is performed at an early stage of tumor culturing with no animal involvement and with minimal tampering with the original tumor cytoarchitecture. This functional precision medicine approach can be exploited in a range of clinical and laboratory settings to improve the clinical management of GB patients and implemented on other cancers as well.

Dissecting the Activity of Catechins as Incomplete Aldose Reductase Differential Inhibitors through Kinetic and Computational Approaches.

The inhibition of aldose reductase is considered as a strategy to counteract the onset of both diabetic complications, upon the block of glucose conversion in the polyol pathway, and inflammation, upon the block of 3-glutathionyl-4-hydroxynonenal reduction. To ameliorate the outcome of aldose reductase inhibition, minimizing the interference with the detoxifying role of the enzyme when acting on toxic aldehydes, "differential inhibitors", i.e., molecules able to inhibit the enzyme depending on the substrate the enzyme is working on, has been proposed. Here we report the characterization of different catechin derivatives as aldose reductase differential inhibitors. The study, conducted through both a kinetic and a computational approach, highlights structural constraints of catechin derivatives relevant in order to affect aldose reductase activity. Gallocatechin gallate and catechin gallate emerged as differential inhibitors of aldose reductase able to preferentially affect aldoses and 3-glutathionyl-4-hydroxynonenal reduction with respect to 4-hydroxynonenal reduction. Moreover, the results highlight how, in the case of aldose reductase, a substrate may affect not only the model of action of an inhibitor, but also the degree of incompleteness of the inhibitory action, thus contributing to differential inhibitory phenomena.

Salivary Proteomics Markers for Preclinical Sjögren's Syndrome: A Pilot Study.

Primary Sjögren's syndrome (pSS) is a complex autoimmune disorder that particularly affects the salivary and lachrymal glands, generally causing a typical dryness of the eyes and of the mouth. The disease encompasses diverse clinical representations and is characterized by B-cell polyclonal activation and autoantibodies production, including anti-Ro/SSA. Recently, it has been suggested that autoantibody profiling may enable researchers to identify susceptible asymptomatic individuals in a pre-disease state. In this pilot study, we used mass spectrometry to analyze and compare the salivary proteomics of patients with established pSS and patients with pre-clinical SS, identifying a common protein signature in their salivary fluid. We found that several inflammatory, immunity-related, and typical acinar proteins (such as MUC5B, PIP, CST4, and lipocalin 1) were differently expressed in pSS and in pre-clinical SSA+ carriers, compared to healthy controls. This suggests that saliva may closely reflect exocrine gland inflammation from the early phases of the disease. This study confirms the value of salivary proteomics for the identification of reliable biomarkers for SS that could be identified, even in a preclinical phase of the disease.

Fluorescence lifetime microscopy unveils the supramolecular organization of liposomal Doxorubicin.

The supramolecular organization of Doxorubicin (DOX) within the standard Doxoves® liposomal formulation (DOX®) is investigated using visible light and phasor approach to fluorescence lifetime imaging (phasor-FLIM). First, the phasor-FLIM signature of DOX® is resolved into the contribution of three co-existing fluorescent species, each with its characteristic mono-exponential lifetime, namely: crystallized DOX (DOXc, 0.2 ns), free DOX (DOXf, 1.0 ns), and DOX bound to the liposomal membrane (DOXb, 4.5 ns). Then, the exact molar fractions of the three species are determined by combining phasor-FLIM with quantitative absorption/fluorescence spectroscopy on DOXc, DOXf, and DOXb pure standards. The final picture on DOX® comprises most of the drug in the crystallized form (∼98%), with the remaining fractions divided between free (∼1.4%) and membrane-bound drug (∼0.7%). Finally, phasor-FLIM in the presence of a DOX dynamic quencher allows us to suggest that DOXf is both encapsulated and non-encapsulated, and that DOXb is present on both liposome-membrane leaflets. We argue that the present experimental protocol can be applied to the investigation of the supramolecular organization of encapsulated luminescent drugs/molecules all the way from the production phase to their state within living matter.

Lysosomal Proteomics Links Disturbances in Lipid Homeostasis and Sphingolipid Metabolism to CLN5 Disease.

CLN5 disease (MIM: 256731) represents a rare late-infantile form of neuronal ceroid lipofuscinosis (NCL), caused by mutations in the CLN5 gene that encodes the CLN5 protein (CLN5p), whose physiological roles stay unanswered. No cure is currently available for CLN5 patients and the opportunities for therapies are lagging. The role of lysosomes in the neuro-pathophysiology of CLN5 disease represents an important topic since lysosomal proteins are directly involved in the primary mechanisms of neuronal injury occurring in various NCL forms. We developed and implemented a lysosome-focused, label-free quantitative proteomics approach, followed by functional validations in both CLN5-knockout neuronal-like cell lines and Cln5-/- mice, to unravel affected pathways and modifying factors involved in this disease scenario. Our results revealed a key role of CLN5p in lipid homeostasis and sphingolipid metabolism and highlighted mutual NCL biomarkers scored with high lysosomal confidence. A newly generated cln5 knockdown zebrafish model recapitulated most of the pathological features seen in NCL disease. To translate the findings from in-vitro and preclinical models to patients, we evaluated whether two FDA-approved drugs promoting autophagy via TFEB activation or inhibition of the glucosylceramide synthase could modulate in-vitro ROS and lipid overproduction, as well as alter the locomotor phenotype in zebrafish. In summary, our data advance the general understanding of disease mechanisms and modifying factors in CLN5 disease, which are recurring in other NCL forms, also stimulating new pharmacological treatments.

Biological Effects of Transforming Growth Factor Beta in Human Cholangiocytes.

TGF-ß is a cytokine implicated in multiple cellular responses, including cell cycle regulation, fibrogenesis, angiogenesis and immune modulation. In response to pro-inflammatory and chemotactic cytokines and growth factors, cholangiocytes prime biliary damage, characteristic of cholangiopathies and pathologies that affect biliary tree. The effects and signaling related to TGF-ß in cholangiocyte remains poorly investigated. In this study, the cellular response of human cholangiocytes to TGF-ß was examined. Wound-healing assay, proliferation assay and cell cycle analyses were used to monitor the changes in cholangiocyte behavior following 24 and 48 h of TGF-ß stimulation. Moreover, proteomic approach was used to identify proteins modulated by TGF-ß treatment. Our study highlighted a reduction in cholangiocyte proliferation and a cell cycle arrest in G0/G1 phase following TGF-ß treatment. Moreover, proteomic analysis allowed the identification of four downregulated proteins (CaM kinase II subunit delta, caveolin-1, NipSnap1 and calumin) involved in Ca2+ homeostasis. Accordingly, Gene Ontology analysis highlighted that the plasma membrane and endoplasmic reticulum are the cellular compartments most affected by TGF-ß. These results suggested that the effects of TGF-ß in human cholangiocytes could be related to an imbalance of intracellular calcium homeostasis. In addition, for the first time, we correlated calumin and NipSnap1 to TGF-ß signaling.