Proteomics Profiling of Heterozygous and Homozygous Patients with ABCA1 Gene Mutation: A Tangier Disease Molecular Map

Tangier Disease (TD) is a rare inherited disorder with approximately 100 worldwide identified cases. Alpha-lipoprotein deficiency is the main characteristic of this disease, associated with a virtual absence of High Density Lipoproteins (HDL) in blood. Additional symptoms are mild hypertriglyceridemia, neuropathy and enlarged, orange-colored tonsils. Genetically TD is caused by mutations in the ABCA1 gene, which prevent the release of cholesterol and phospholipids from cells, leading to the accumulation of lipids within cells and body tissues. In this work a TD patient and his parental heterozygous were examined from a proteomics point of view. Plasma as well as proteome and secretome of circulating monocytes were analyzed. Plasma proteins underlined in TD the imbalance of lipid trafficking and metabolism, associated with the stimulation of pro-inflammatory pathways. Proteome and secretome of monocytes highlighted an extensive down regulation of mitochondrial enzymes and vesicular trafficking agents along with a substantial cytoskeletal rearrangement, suggesting a reduced activation state of monocytes from TD homozygous patient. This work is the first proteomics profiling of heterozygous and homozygous TD phenotypes and it suggests a TD case as a model to understand general mechanisms of lipid transport and metabolism and their linkage to inflammatory processes

Reactivity of Heteropolytungstate and Heteropolymolybdate Metal Transition Salts in the Synthesis of Dimethyl Carbonate from Methanol and CO2

A series of Keggin-type heteropoly compounds (HPC) having different countercations (Co, Fe) and different addenda atoms (W, Mo) were synthesized and characterized by means of Fourier-Transform Infrared Spectrometer (FT-IR) and X-ray powder diffraction (XRD). The catalytic properties of the prepared catalysts for the dimethyl carbonate (DMC) synthesis from CO2 and CH3OH were investigated. The experimental results showed that the catalytic activity is significantly influenced by the type of the countercation and addenda atoms transition metal. Among the catalysts examined, Co1.5PW12O40 is the most active for the DMC synthesis, owing to the synergetic effect between Co and W. Investigating the effect of the support showed that the least acidic one (Al2O3) enhanced the conversion but decreased the DMC selectivity in favor of that of methyl formate (MF), while that of dimethoxy methane remained stable

Proteomic Modulation in TGF-β-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-  (TGF- )-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- . Moreover, curcumin nanoformulation positively influenced the expression of several proteins involved in TGF- -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

A proteomics approach to the study of bleomycin- induced lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most severe lung fibrotic form and very few pharmacological therapies are available at present. Key events in the onset of the disease are the activation of fibroblasts to myofibroblasts and the production and release of extracellular matrix (ECM) and molecular factors. Primary murine lung fibroblasts were isolated and their activation induced by Bleomycin (BLM) treatment. Extracellular Vesicles (EV) were isolated and protein extracted. Released soluble proteins (Secretome) and EV-derived proteins were reduced, alkylated and trypsin digested. A nano-LC-MS/MS SWATHTM approach was used for the proteomics analyses. Specific proteins with a putative role in the transition from physiological to fibrotic conditions, such as several matrix metalloproteinases (MMPs), osteopontin (OPN), chitinase-3-like protein1 (CHI3L1) and CD44 resulted differentially released from BLM-treated fibroblasts as compared with untreated lung fibroblasts. Our results provide further understanding of the pathophysiological features of lung fibrosis, and suggest specific target for pharmacological treatments

Lipid biomarkers in statin users with coronary artery disease annotated by coronary computed tomography angiography

Molecular markers are suggested to improve the diagnostic and prognostic accuracy in patients with coronary artery disease (CAD) beyond current clinical scores based on age, gender, symptoms and traditional risk factors. In this context, plasma lipids are emerging as predictors of both plaque composition and risk of future events. We aim to identify plasma lipid biomarkers associated to CAD indexes of stenosis severity, plaque lipid content and a comprensive score of CAD extent and its risk. We used a simple high performance liquid chromatography-tandem mass spectrometry method to identify 69 plasma lipids in 132 subjects referred to Coronary Computed Tomography Angiography (CCTA) for suspected CAD, all under statin treatment. Patients were stratified in groups using three different CCTA-based annotations: CTA-risk score, lipid plaque prevalence (LPP) ratio and the coronary artery disease-reporting and data system (CAD-RADS). We identified a common set of lipid biomarkers composed of 7 sphingomyelins and 3 phosphatidylethanolamines, which discriminates between high risk CAD patients and controls regardless of the CAD annotations used (CTA score, LPP ratio, or CAD-RADS). These results highlight the potential of circulating lipids as biomarkers of stenosis severity, non calcified plaque composition and overall plaque risk of events.Cardiolog

Inflammatory and antioxidant pattern unbalance in "clopidogrel-resistant" patients during acute coronary syndrome.

Background. In acute coronary syndrome (ACS), inflammation and redox response are associated with increased residual platelet reactivity (RPR) on clopidogrel therapy. We investigated whether clopidogrel interaction affects platelet function and modulates factors related to inflammation and oxidation in ACS patients differently responding to clopidogrel. Material andMethods. Platelet aggregation was measured in 29 ACS patients on dual (aspirin/clopidogrel) antiplatelet therapy. Nonresponders (NR) were defined as RPR ≥70% by ADP. Several inflammatory and redox parameters were assayed and platelet proteome was determined. Results. Eight (28%) out of 29 ACS patients resulted NR to clopidogrel. At 24 hours, the levels of Th2-type cytokines IL-4, IFN, andMCP-1 were higher in NR, while blood GSH (r-GSHbl) levels were lower in NR than responders (R). Proteomic analysis evidenced an upregulated level of platelet adhesion molecule, CD226, and a downregulation of the antioxidant peroxiredoxin-4. In R patients the proinflammatory cytokine IL-6 decreased, while the anti-inflammatory cytokine IL-1Ra increased. Conclusions. In patients with high RPR on clopidogrel therapy, an unbalance of inflammatory factors, platelet adhesion molecules, and circulatory and platelet antioxidantmolecules was observed during the acute phase. Proinflammatory milieu persists in nonresponders for a long time after the acute event while antioxidant blood factors tend to conform to normal responsiveness

RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells.

BACKGROUND: Embryonic stem cells are intrinsically unstable and differentiate spontaneously if they are not shielded from external stimuli. Although the nature of such instability is still controversial, growing evidence suggests that protein translation control may play a crucial role. RESULTS: We performed an integrated analysis of RNA and proteins at the transition between naïve embryonic stem cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators are specifically released from translational inhibition mediated by RNA-induced silencing complex (RISC). This suggests that, prior to differentiation, the propensity of embryonic stem cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators is reinstated following acute inactivation of RISC and it correlates with loss of stemness markers and activation of early cell differentiation markers in treated embryonic stem cells. CONCLUSIONS: We propose that RISC-mediated inhibition of specific sets of chromatin regulators is a primary mechanism for preserving embryonic stem cell pluripotency while inhibiting the onset of embryonic developmental programs.This work was funded by: FIRB RBAP10L8TY (MIUR), Fondazione Roma and PAINCAGE FP7 Collaborative Project number 603191 (RB,MD); Flagship Project InterOmics PB.05 and MIUR-PRIN-2012 (FC); Wellcome Trust Core Grant reference 092096 and Cancer Research UK Grant Reference C6946/A14492 (LP); CRUK-Cambridge Institute Core Grant reference C14303/A17197 (DB)

The landscape of BRAF transcript and protein variants in human cancer

Background: The BRAF protein kinase is widely studied as a cancer driver and therapeutic target. However, the regulation of its expression is not completely understood. Results: Taking advantage of the RNA-seq data of more than 4800 patients belonging to 9 different cancer types, we show that BRAF mRNA exists as a pool of 3 isoforms (reference BRAF, BRAF-X1, and BRAF-X2) that differ in the last part of their coding sequences, as well as in the length (BRAF-ref: 76 nt; BRAF-X1 and BRAF-X2: up to 7 kb) and in the sequence of their 3'UTRs. The expression levels of BRAF-ref and BRAF-X1/X2 are inversely correlated, while the most prevalent among the three isoforms varies from cancer type to cancer type. In melanoma cells, the X1 isoform is expressed at the highest level in both therapy-naïve cells and cells with acquired resistance to vemurafenib driven by BRAF gene amplification or expression of the Δ[3-10] splicing variant. In addition to the BRAF-ref protein, the BRAF-X1 protein (the full length as well as the Δ[3-10] variant) is also translated. The expression levels of the BRAF-ref and BRAF-X1 proteins are similar, and together they account for BRAF functional activities. In contrast, the endogenous BRAF-X2 protein is hard to detect because the C-terminal domain is selectively recognized by the ubiquitin-proteasome pathway and targeted for degradation. Conclusions: By shedding light on the repertoire of BRAF mRNA and protein variants, and on the complex regulation of their expression, our work paves the way to a deeper understanding of a crucially important player in human cancer and to a more informed development of new therapeutic strategies

Effects of Intracoronary Alteplase on Microvascular Function in Acute Myocardial Infarction

Background—Impaired microcirculatory reperfusion worsens prognosis following acute ST‐segment–elevation myocardial infarction. In the T‐TIME (A Trial of Low‐Dose Adjunctive Alteplase During Primary PCI) trial, microvascular obstruction on cardiovascular magnetic resonance imaging did not differ with adjunctive, low‐dose, intracoronary alteplase (10 or 20 mg) versus placebo during primary percutaneous coronary intervention. We evaluated the effects of intracoronary alteplase, during primary percutaneous coronary intervention, on the index of microcirculatory resistance, coronary flow reserve, and resistive reserve ratio. Methods and Results—A prespecified physiology substudy of the T‐TIME trial. From 2016 to 2017, patients with ST‐segment–elevation myocardial infarction ≤6 hours from symptom onset were randomized in a double‐blind study to receive alteplase 20 mg, alteplase 10 mg, or placebo infused into the culprit artery postreperfusion, but prestenting. Index of microcirculatory resistance, coronary flow reserve, and resistive reserve ratio were measured after percutaneous coronary intervention. Cardiovascular magnetic resonance was performed at 2 to 7 days and 3 months. Analyses in relation to ischemic time (<2, 2–4, and ≥4 hours) were prespecified. One hundred forty‐four patients (mean age, 59±11 years; 80% male) were prospectively enrolled, representing 33% of the overall population (n=440). Overall, index of microcirculatory resistance (median, 29.5; interquartile range, 17.0–55.0), coronary flow reserve(1.4 [1.1–2.0]), and resistive reserve ratio (1.7 [1.3–2.3]) at the end of percutaneous coronary intervention did not differ between treatment groups. Interactions were observed between ischemic time and alteplase for coronary flow reserve (P=0.013), resistive reserve ratio (P=0.026), and microvascular obstruction (P=0.022), but not index of microcirculatory resistance. Conclusions—In ST‐segment–elevation myocardial infarction with ischemic time ≤6 hours, there was overall no difference in microvascular function with alteplase versus placebo