Proteomic Characterization of Collagen-Based Animal Glues for Restoration.

Animal glues are widely used in restoration as adhesives, binders, and consolidants for organic and inorganic materials. Their variable performances are intrinsically linked to the adhesive properties of collagen, which determine the chemical, physical, and mechanical properties of the glue. We have molecularly characterized the protein components of a range of homemade and commercial glues using mass spectrometry techniques. A shotgun proteomic analysis provided animal origin, even when blended, and allowed us to distinguish between hide and bone glue on the basis of the presence of collagen type III, which is abundant in connective skin/leather tissues and poorly synthetized in bones. Furthermore, chemical modifications, a consequence of the preparation protocols from the original animal tissue, were thoroughly evaluated. Deamidation, methionine oxidation, and backbone cleavage have been analyzed as major collagen modifications, demonstrating their variability among different glues and showing that, on average, bone glues are less deamidated than hide glues, but more fragmented, and mixed-collagen glues are overall less deamidated than pure glues. We believe that these data may be of general analytical interest in the characterization of collagen-based materials and may help restorers in the selection of the most appropriate materials to be used in conservation treatments.

Molecular signatures written in bone proteins of 79 AD victims from Herculaneum and Pompeii.

An extensive proteomic analysis was performed on a set of 12 bones of human victims of the eruption that in AD 79 rapidly buried Pompeii and Herculaneum, allowing the detection of molecular signatures imprinted in the surviving protein components. Bone collagen survived the heat of the eruption, bearing a piece of individual biological history encoded in chemical modifications. Here we show that the human bone proteomes from Pompeii are more degraded than those from the inhabitants of Herculaneum, despite the latter were exposed to temperatures much higher than those experienced in Pompeii. The analysis of the specimens from Pompeii shows lower content of non-collagenous proteins, higher deamidation level and higher extent of collagen modification. In Pompeii, the slow decomposition of victims' soft tissues in the natural dry-wet hydrogeological soil cycles damaged their bone proteome more than what was experienced at Herculaneum by the rapid vanishing of body tissues from intense heat, under the environmental condition of a permanent waterlogged burial context. Results herein presented are the first proteomic analyses of bones exposed to eruptive conditions, but also delivered encouraging results for potential biomarkers that might also impact future development of forensic bone proteomics.

Targeted Metabolomics: The LC-MS/MS Based Quantification of the Metabolites Involved in the Methylation Biochemical Pathways.

Biochemical methylation reactions mediate the transfer of the methyl group regulating vital biochemical reactions implicated in various diseases as well as the methylation of DNA regulating the replication processes occurring in living organisms. As a finite number of methyl carriers are involved in the methyl transfer, their quantification could aid towards the assessment of an organism's methylation potential. An Hydrophilic Interaction Chromatography-Liquid Chromatography Multiple Reaction Monitoring (HILIC-LC-MRM) mass spectrometry (MS) methodology was developed and validated according to Food & Drug Administration (FDA), European Medicines Agency (EMA), and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) for the simultaneous determination of nine metabolites i.e., B12, folic acid, 5-methyltetrahydrofolate, S-adenosylmethionine, S-adenosylhomocysteine, betaine, phosphocholine, N,N-dimethylglycine, and deoxythymidine monophosphate in human blood plasma. The sample pretreatment was based on a single step Solid-phase extraction (SPE) methodology using C18 cartridges. The methodology was found to accurately quantitate the analytes under investigation according to the corresponding dynamic range proposed in the literature for each analyte. The applicability of the method was assessed using blood donor samples and its applicability demonstrated by the assessment of their basal levels, which were shown to agree with the established basal levels. The methodology can be used for diagnostic purposes as well as for epigenetic screening.

Optimization of Inulin Hydrolysis by Penicillium lanosocoeruleum Inulinases and Efficient Conversion Into Polyhydroxyalkanoates.

Inulin, a polydisperse fructan found as a common storage polysaccharide in the roots of several plants, represents a renewable non-food biomass resource for the synthesis of bio-based products. Exploitation of inulin-containing feedstocks requires the integration of different processes, including inulinase production, saccharification of inulin, and microbial fermentation for the conversion of released sugars into added-value products. In this work paper, a new microbial source of inulinase, Penicillium lanosocoeruleum, was identified through the screening of a fungal library. Inulinase production using inulin as C-source was optimized, reaching up to 28 U mL-1 at the 4th day of growth. The fungal inulinase mixture (PlaI) was characterized for pH and temperature stability and activity profile, and its isoenzymes composition was investigated by proteomic strategies. Statistical optimization of inulin hydrolysis was performed using a central composite rotatable design (CCRD), by analyzing the effect of four factors. In the optimized conditions (T, 45.5°C; pH, 5.1; substrate concentration, 60 g L-1; enzyme loading, 50 U gsubstrate -1), up to 96% inulin is converted in fructose within 20 h. The integration of PlaI in a process for polyhydroxyalkanoate (PHA) production by Cupriavidus necator from inulin was tested in both separated hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). A maximum of 3.2 g L-1 of PHB accumulation, corresponding to 82% polymer content, was achieved in the SSF. The proved efficiency in inulin hydrolysis and its effective integration into a SSF process pave the way to a profitable exploitation of the PlaI enzymatic mixture in inulin-based biorefineries.

A versatile and user-friendly approach for the analysis of proteins in ancient and historical objects.

Identification and characterization of ancient proteins still require technical developments towards non-invasiveness, sensitivity, versatility and ease of use of the analyses. We report that the enzyme functionalized films, described in Cicatiello et al. (2018), can be used efficiently on the surface of different objects ranging from fixative-coated paper to canvas to the coating on an albumen photograph, as well as the much harder surfaces of ivory objects and the proteinaceous binders in the decoration of a wooden Egyptian coffin. The mixture of digested peptides that are efficiently captured on the functionalized surface are also amenable to LC-MS/MS analysis, which is necessary to confidently identify chemical modifications induced upon degradation, in order to characterize the conservation state of proteins. Moreover, in a two-step procedure, we have combined the trypsin functionalized film with a PNGaseF functionalized film, which adds a deglycosylation pretreatment allowing improved detection of glycosylated proteins. SIGNIFICANCE: User friendly trypsin functionalized films were implemented to expand their potential as versatile, modular tools that can be widely exploited in the world of diagnosis of cultural heritage objects, ancient proteins, and palaeoproteomics: a procedure that could be carried out by conservators or archaeologists first on-site and later analysed with standard MS techniques.

Minimally Invasive and Portable Method for the Identification of Proteins in Ancient Paintings.

A novel method for the analysis of proteinaceous materials present on painted surfaces was developed by taking advantage of the adhesive ability of some fungal proteins which can form a stable and homogeneous layer on flexible transparency sheets able to capture trypsin in a fully active form. We demonstrated that the bioactive sheets were able to efficiently digest proteins, present as such, on surfaces of painted tests and historical samples, releasing peptides that can allow an easy and confident identification of the proteinaceous binders by standard bottom-up proteomic approach. By this method there is no need: (i) to transport the artifacts and (ii) to remove, even at micro level, a sample from the object. The ingenuity of the method lies in the easily accommodated sampling coupled with a minimal invasiveness.

Evolved Gas Analysis-Mass Spectrometry to Identify the Earliest Organic Binder in Aegean Style Wall Paintings.

An organic binder was identified in the painted fragments from the Canaanite palace of Tel Kabri, Israel. Recently dated to the late 18th century B.C.E. by 14 C, Tel Kabri is the most ancient of the Eastern Mediterranean sites in which Aegean style paintings have been found. The application of pigments was suspected to be using an organic binding medium, particularly for the Egyptian Blue pigment. Samples of blue paint were examined using evolved gas analysis-mass spectrometry (EGA-MS) in order to overcome the analytical challenges imposed by highly degraded aged proteinaceous materials. Egg was identified as the binder based on the presence of hexadecanonitrile and octadecanonitrile, confirming the use of a secco painting technique. Lysozyme C from Gallus gallus was detected by proteomics analysis, confirming the presence of egg. To our knowledge, this is the earliest use of egg as a binder in Aegean style wall paintings.

Roasting has a distinct effect on the antimutagenic activity of coffee varieties.

Coffee is a highly consumed beverage throughout the world. Its popularity derives from its organoleptic properties that are a result of the roasting process. Roasting greatly alters a coffee bean's composition and possibly its bioactivity. In the current study, green as well as roasted extracts from both Coffea arabica (Brazil and Decaf) and Coffea canephora (Robusta) species were tested for their antimutagenic activity using the Ames test. In addition, a compositional analysis was conducted to identify the main components, mainly Chlorogenic acid isomers (CGA) and derivatives present in the extracts using UHPLC-ESI(±) and HRMS/MS methods According to the results, all extracts exhibited strong antimutagenic activity against the oxidizing factor tert-Butyl hydroperoxide, a Reactive Oxygen Species-producing compound. Roasting had a distinct effect on the antimutagenic activity of coffee, enhancing it in the Brazil variety and having no effect in the Decaf and Robusta varieties. In addition, all coffee extracts exhibited reducing activity as well as the ability to scavenge (albeit differentially) both the superoxide and hydroxyl radicals, implying that their potential antimutagenic effect can be partially attributed to their free radical scavenging activity.

Roasted and green coffee extracts show antioxidant and cytotoxic activity in myoblast and endothelial cell lines in a cell specific manner.

Coffee is one of the most highly consumed beverages with potential beneficial health implications, however its molecular mechanism of action has not been completely elucidated yet. To that cause, the polyphenolic composition of different coffee extracts (from Light, Medium and Dark roasts as well as green beans) was examined by UHPLC-HRMS analysis, indicating chlorogenic acids isomers as the main constituents. In the following step, the toxicity of the extracts was tested in myoblasts and endothelial cells and differential toxicity of green and roasted samples was displayed as the myoblasts were more sensitive to green coffee extracts, in contrast to the endothelial cells. Subsequently, biologically relevant, non-cytotoxic extract concentrations were administered to explore their potential effect on cell redox status using flow cytometry and spectrophotometric assays. The results indicated that all coffee extracts improved cell redox status, however differences were observed between the two different cell lines tested, implying that coffee compounds display cell- and tissue-specificity. Glutathione levels were increased in almost all cases up to 70%, while the roasting degree affected the free radical scavenging potential of the extracts and their ability to protect from macromolecular oxidation as exhibited by the differences in ROS, CARB and TBARS levels, especially in the myoblasts.

Effect of polyphenols from coffee and grape on gene expression in myoblasts.

Coffee and grape contain various bioactive compounds like polyphenols that may exert beneficial effects, especially antioxidant activity, on human health upon consumption. However, the molecular mechanisms through which these effects are achieved are not fully elucidated. Thus, in the present study in order to investigate these mechanisms, a whole genome expression DNA microarray analysis was carried out in myoblasts treated with polyphenols of coffee and grape pomace at concentrations that improved the redox status. Grape was composed of catechin, epicatechin, cyanidin, malvidin, delphinidin, petunidin, myrtillin, kuromanin, oenin, peonidin, quercetin, gallic acid and caftaric acid as LC-MS revealed, with a total polyphenolic content (TPC) of 648 mg of gallic acid equivalents/g of dry matter. Coffee had a TPC of 42.61 mg GAE/g coffee and was composed of 3-chlorogenic acid (16.61 mg/g), 4- and 5-chlorogenic acids (13.62 mg/g), as UHPLC-HRMS revealed. According to the results, grape polyphenols altered mainly the expression of cytoskeleton and differentiation-associated genes, while coffee compounds had a more profound effect, on the expression levels of many metabolic and antioxidant genes possibly through the nuclear factor (erythroid-derived 2) like-2 (Nrf2) pathway.

Enhancement of Antioxidant Mechanisms and Reduction of Oxidative Stress in Chickens after the Administration of Drinking Water Enriched with Polyphenolic Powder from Olive Mill Waste Waters.

The aim of the study was to examine the effects of a polyphenolic powder from olive mill wastewater (OMWW) administered through drinking water, on chickens' redox status. Thus, 75 chickens were divided into three groups. Group A was given just drinking water, while groups B and C were given drinking water containing 20 and 50 µg/ml of polyphenols, respectively, for 45 days. The antioxidant effects of the polyphenolic powder were assessed by measuring oxidative stress biomarkers in blood after 25 and 45 days of treatment. These markers were total antioxidant capacity (TAC), protein carbonyls (CARB), thiobarbituric acid reactive species (TBARS) and superoxide dismutase activity (SOD) in plasma, and glutathione (GSH) and catalase activity in erythrocytes. The results showed that CARB and TBARS were decreased significantly in groups B and C, and SOD decreased in group B compared to that in group A. TAC was increased significantly in group C and GSH was increased in group B, while catalase activity was increased in groups B and C compared to that in group A. In conclusion, this is the first study showing that supplementation of chickens with polyphenols from OMWW through drinking water enhanced their antioxidant mechanisms and reduced oxidative stress-induced damage.