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Evidence is rapidly growing regarding a role of astroglial cells in the pathogenesis of Alzheimer’s disease (AD), and the hippocampus is one of the important brain regions affected in AD. While primary astroglial cultures, both from wild-type mice and from rodent models of AD, have been useful for studying astrocyte-specific alterations, the limited cell number and short primary culture lifetime have limited the use of primary hippocampal astrocytes. To overcome these limitations, we have now established immortalized astroglial cell lines from the hippocampus of 3xTg-AD and wild-type control mice (3Tg-iAstro and WT-iAstro, respectively). Both 3Tg-iAstro and WT-iAstro maintain an astroglial phenotype and markers (glutamine synthetase, aldehyde dehydrogenase 1 family member L1 and aquaporin-4) but display proliferative potential until at least passage 25. Furthermore, these cell lines maintain the potassium inward rectifying (Kir) current and present transcriptional and proteomic profiles compatible with primary astrocytes. Importantly, differences between the 3Tg-iAstro and WT-iAstro cell lines in terms of calcium signaling and in terms of transcriptional changes can be re-conducted to the changes previously reported in primary astroglial cells. To illustrate the versatility of this model we performed shotgun mass spectrometry proteomic analysis and found that proteins related to RNA binding and ribosome are differentially expressed in 3Tg-iAstro vs WT-iAstro. In summary, we present here immortalized hippocampal astrocytes from WT and 3xTg-AD mice that might be a useful model to speed up research on the role of astrocytes in AD.

We recently developed a new method that uses a functionalized film, which non-invasively extracts the proteins from different surfaces, for the identification of proteinaceous materials or binders from precious and ancient artworks. In this research, we demonstrate the efficacy of the method on varnished wood panels and canvases. We also show the application of this method to several historical samples. In particular, we discuss the results obtained from the analysis of: a polychrome wooden altarpiece of the XIV century, a polychrome sandstone capital, a rare polychrome alabaster, several wood medieval and renaissance panels (Pietro Gallo, Jean Bapteur, Defendente Ferrari, Martino Spanzotti, Gerolamo Giovenone) and a detached fresco by Antoine de Lonhy. Through the use of this method we were able to identify the binders from a rare sample of painted leather casket from a Parisian manufactory (beginning of XIV century) and from a Tuscan wooden reliquary. Moreover, by adopting the quantitative analysis, we discovered different artistic techniques used to paint two different areas of an “a secco” painting from Reggia di Colorno (Italy). In conclusion, this study reports the first use of this non-invasive method for the characterization of proteins from a wide range of ancient objects belonging to the world Cultural Heritage without the need to transport or micro-invasively sampling the artifacts.

Physical activity improves overall health and counteracts metabolic pathologies. Adipose tissue and bone are important key targets of exercise; the prevalence of diseases associated with suboptimal physical activity levels has increased in recent times as a result of lifestyle changes. Mesenchymal stem cells (MSCs) differentiation in either osteogenic or adipogenic lineage is regulated by many factors. Particularly, the expression of master genes such as RUNX2 and PPARγ2 is essential for MSC commitment to osteogenic or adipogenic differentiation, respectively. Besides various positive effects on health, some authors have reported stressful outcomes as a consequence of endurance in physical activity. We looked for further clues about MSCs differentiation and serum proteins modulation studying the effects of half marathon in runners by means of gene expression analyses and a proteomic approach. Our results demonstrated an increase in osteogenic commitment and a reduction in adipogenic commitment of MSCs. In addition, for the first time we have analyzed the proteomic profile changes in runners after half-marathon activity in order to survey the related systemic adjustments. The shotgun proteomic approach, performed through the immuno-depletion of the 14 most abundant serum proteins, allowed the identification of 23 modulated proteins after the half marathon. Interestingly, proteomic data showed the activation of both inflammatory response and detoxification process. Moreover, the involvement of pathways associated to immune response, lipid transport and coagulation, was elicited. Notably, positive and negative effects may be strictly linked. Data are available via ProteomeXchange with identifier PXD006704. Significance We describe gene expression and proteomic studies aiming to an in-depth understanding of half-marathon effects on bone and adipogenic differentiation as well as biological phenomena involved in sport activity. We believe that this novel approach suggests the physical effects on overall health and show the different pathways involved during half marathon. Contents of the paper have not been published or submitted for publication elsewhere. The authors declare no conflict of interest.

Purpose The present research reports the study the of plasma proteome profile of stable coronary artery disease (CAD) patients characterized by different levels of total Apolipoprotein-CIII (Apo C-III), a prognostic marker for cardiovascular risk. Experimental design Two subgroups of CAD patients (n = 52) with divergent concentrations of total circulating Apo C-III (≤ and ≥10 mg dL-1 ) are examined using a shotgun proteomic approach. Validation experiments are also performed with immunochemistry methods including both the patients affected by CAD (n = 119) and the subjects without CAD (CAD-free; n = 58). Results are analyzed by bioinformatics tools and multivariate statistics. Results A total of 188 proteins are quantified among the patients. The fold change analysis and the partial least square discriminant analysis show a clear separation of the two groups. Lipoproteins (Apo C-II and Apo E), retinol-binding protein 4, and vitronectin are upregulated in patients with high Apo C-III, while alpha-1 antitrypsin is downregulated. Conclusions and clinical relevance In this pilot study, the differential expression of plasma proteins related to different concentrations of Apo C-III is defined, suggesting possible new players involved in the Apo C-III-associated process of arterial damage. Data are available via ProteomeXchange with identifier PXD005973.

The diagnosis and management of Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is still challenging. There is no definitive gold standard diagnostic test, which is made on patient history and with endoscopic and histological findings. This study analyzed serum proteins and fatty acids using mass spectrometry-based techniques. Quantitation of serum proteins was performed by depleting 14 high-abundance proteins, followed by tryptic digestion and LC-MS analysis, while fatty acids were analyzed using GC-MS. Bioinformatic tools were used to identify several new potential biomarkers for an early and non-invasive diagnosis of IBD, and to differentiate CD from UC. Moreover, the diagnostic power of the MS-identified biomarkers was also corroborated by Western Blot and ELISA assays. Hence, we identified the biological functions and pathways involved in the various subsets of IBD. Coagulation, fibrinolysis and acute phase response processes were found to be strongly involved in the condition. The involvement of several fatty acids, such as anti-inflammatory mediators, was also identified. Finally, proteomic and lipidomic data were integrated by using combinatorial and multivariate analyses to discover new combined biomarkers and to study the molecular pathways involved in IBD.

Background: Apolipoprotein C-III (ApoC-III), a key regulator of plasma triglyceride (TG), is present in three isoforms, i.e. non-sialylated (ApoC-III0), monosialylated (ApoC-III1) and disialylated (ApoC-III2). We aimed at quantifying the distribution of the ApoC-III glycoforms in patients with angiographically demonstrated coronary artery disease (CAD) according to levels of total ApoC-III plasma concentration. Methods: ApoC-III glycoforms were quantified by a specifically developed, high-resolution, mass spectrometry method in unrelated CAD patients. Lipoprotein lipase (LPL) activity was estimated by a fluorescence-based method. Results: In 101 statin-treated CAD patients, the absolute concentrations of the three glycoforms similarly increased across ApoC-III quartiles, but the proportion of ApoC-III1 rose whereas that of ApoC-III0 decreased progressively by increasing total ApoC-III concentrations. The proportion of ApoC-III2 was quite constant throughout the whole range of total ApoC-III. A higher proportion of ApoC-III1 reflected an unfavorable lipid profile characterized by high levels of TG, total and low density lipoprotein cholesterol, ApoE and reduced ApoA-I. The correlations between ApoC-III glycoforms and TG were confirmed in 50 statin-free CAD patients. High concentration of total ApoC-III was associated with low LPL activity, while no correlation was found for the relative proportion of glycoforms. Conclusions: Specific patterns of ApoC-III glycoforms are present across different total ApoC-III concentrations in CAD patients. The inhibitory effect of ApoC-III on LPL appears related to total ApoC-III concentration, but not to the relative proportion of ApoC-III glycoforms.

We conducted a proteomics study in order to detect the proteomic method which provides the most complete characterization of the proteins of rice milk. In particular, we compared the results obtained from LC-MS/MS after protein precipitation with acetone or TCA, as well as the results obtained from LC-MS/MS after protein prefractionation based on SDS-PAGE (GeLC-MS/MS) or ProteoMiner™ technology (ProteoMiner-LC-MS/MS), and after peptide prefractionation based on IEF (pIEF-LC-MS/MS). A total of 158 protein species have been detect in rice milk. The physical-chemical analysis and classification of the identified proteins were also reported. In particular, we showed that pIEF-LC-MS/MS method led to a significant increase in the proteome coverage, allowing the identification of a total of 96 proteins of milk rice. This study demonstrates the utility of a prefractionation step based on pIEF before the shotgun proteomic analysis and offers an in-depth insight into the rice milk proteome.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal of all human cancers with a high mortality rate. Resistance to conventional treatments and chemotherapeutics is a typical feature of PDAC. To investigate the causes of drug resistance it is essential to deeply investigate the mechanism of action of chemotherapeutics. In this study, we performed an in depth shotgun proteomic approach using the label-free proteomic SWATH-MS analysis to investigate novel insights of the mechanism of action of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) in PDAC cells. This proteomic analysis in PaCa44 cells and data elaboration of TSA-regulated proteins by bioinformatics showed an overall up-regulation of cytokeratins and other proteins related to the cytoskeleton organization, keratinization, and apoptotic cell death. On the contrary, a large amount of the down-regulated proteins by TSA treatment belongs to the cellular energetic metabolism and to the machinery of protein synthesis, such as ribosomal proteins, determining synergistic cell growth inhibition by the combined treatment of TSA and the glycolytic inhibitor 2-deoxy-d-glucose in a panel of PDAC cell lines. Data are available via ProteomeXchange with identifier PXD007801.

Proteins and small molecules from ancient objects and cultural heritage can provide key information and contribute to study the context of objects and artists. However, all present-day protocols and strategies for the analysis of ancient samples are often invasive and require microsampling. Here, we present a new method for the noninvasive analysis of proteins and small molecules: the technique uses a special ethyl-vinyl acetate film functionalized with strong cation/anion exchange and C8 resins, for interacting with both proteins and small molecules present on the surface of the objects, followed by LC-MS/MS analysis. The new method was fully validated for the determination of both proteins and small molecules on several types of supports, showing excellent analytical performances such as, for example, R2 of the calibration curve of 0.98 and 0.99 for proteins and small molecules, low but very repeatable recoveries, particularly adequate for investigations on precious ancient samples that must not be altered by the analytical procedure. ESEM images and LED multispectral imaging confirmed that no damages or alterations occurred onto the support surfaces and no residues were left from the extractive film. Finally, the new method was applied for the characterization of the binders of a historical fresco of the XVI century from the Flemish painter Paul Brill and of a recently discovered fresco from Isidoro Bianchi (XVII century). Moreover the method was employed for the identification of the colorant used by Pietro Gallo (XIV century) on a wood panel. The method here reported can be easily applied to any other research on ancient precious objects and cultural heritage, since it does not require microsampling and the proteins/small molecules extraction can be performed directly in situ, leaving the object unchanged and intact. (Figure Presented)