Publications by authors named "Vera Muccilli"

55 Publications

Quantitative Label-Free Comparison of the Metabolic Protein Fraction in Old and Modern Italian Wheat Genotypes by a Shotgun Approach.

Molecules 2021 Apr 29;26(9). Epub 2021 Apr 29.

Laboratory of Organic Mass Spectrometry, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

Wheat represents one of the most important cereals for mankind. However, since wheat proteins are also the causative agent of several adverse reactions, during the last decades, consumers have shown an increasing interest in the old wheat genotypes, which are generally perceived as more "natural" and healthier than the modern ones. Comparison of nutritional value for modern and old wheat genotypes is still controversial, and to evaluate the real impact of these foods on human health comparative experiments involving old and modern genotypes are desirable. The nutritional quality of grain is correlated with its proteomic composition that depends on the interplay between the genetic characteristics of the plant and external factors related to the environment. We report here the label-free shotgun quantitative comparison of the metabolic protein fractions of two old Sicilian landraces (Russello and Timilia) and the modern variety Simeto, from the 2010-2011 and 2011-2012 growing seasons. The overall results show that Timilia presents the major differences with respect to the other two genotypes investigated. These differences may be related to different defense mechanisms and some other peculiar properties of these genotypes. On the other hand, our results confirm previous results leading to the conclusion that with respect to a nutritional value evaluation, there is a substantial equivalence between old and modern wheat genotypes. Data are available via ProteomeXchange with identifier .
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http://dx.doi.org/10.3390/molecules26092596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8124627PMC
April 2021

Nanosponges based on self-assembled starfish-shaped cucurbit[6]urils functionalized with imidazolium arms.

Chem Commun (Camb) 2021 Apr 16;57(30):3664-3667. Epub 2021 Mar 16.

Dipartimento di Scienze del Farmaco e della Salute, Università di Catania, V.le A. Doria, 95125-Catania, Italy.

A new porous material based on the first supramolecular cucurbituril-based nanosponge was synthesized by the functionalization of cucurbit[6]uril with twelve 1-(2-bromoethyl)-3-methyl-1H-imidazol-3-ium arms. The porous structure and the high adsorption capacity were demonstrated through surface area measurements and carbon dioxide adsorption. The new supramolecular sponge showed attractive properties such as (i) a highly porous structure that allowed CO capture, (ii) the possibility to reuse the adsorbed CO for organic synthesis, and (iii) an exciting thermal stability up to around 800 °C, with the potential use of this material in high temperature reactions. Finally, the reuse of CO was successfully investigated in the carboxylation reaction of phenylacetylene.
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http://dx.doi.org/10.1039/d1cc00990gDOI Listing
April 2021

Natural Isoflavones and Semisynthetic Derivatives as Pancreatic Lipase Inhibitors.

J Nat Prod 2021 03 1;84(3):654-665. Epub 2021 Mar 1.

Dipartimento di Scienze Chimiche, Università degli Studi di Catania, V.le A. Doria 6, 95125 Catania, Italy.

Obesity, now widespread all over the world, is frequently associated with some chronic diseases. Thus, there is a growing interest in the prevention and treatment of obesity. To date, the only antiobesity drug is orlistat, a natural product-derived pancreatic lipase (PL) inhibitor with some undesired side effects. In the last decades, many natural compounds or derivatives have been evaluated as potential PL inhibitors, and natural polyphenols are among the most promising for possible exploitation as antiobesity agents. However, few studies have been devoted to isoflavones. In this work, we report a study on the PL inhibitory properties of a small library of semisynthetic isoflavone derivatives together with the natural leads daidzein (), genistein (), and formononetin (). In vitro lipase inhibition assay showed that is the most promising PL inhibitor. Among synthetic isoflavones, the hydroxylated and brominated derivatives were more potent than their natural leads. Detailed studies through fluorescence measurements and kinetics of lipase inhibition showed that and the bromoderivatives and have the greatest affinity for PL. Docking studies corroborated these findings highlighting the interactions between isoflavones and the enzyme, confirming that hydroxylation and bromination are useful modifications.
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http://dx.doi.org/10.1021/acs.jnatprod.0c01387DOI Listing
March 2021

Synthesis, DNA/RNA-interaction and biological activity of benzo[k,l]xanthene lignans.

Bioorg Chem 2020 11 26;104:104190. Epub 2020 Aug 26.

Ruđer Bošković Institute, Division of Organic Chemistry and Biochemistry, Bijenička cesta 54, 10000 Zagreb, Croatia. Electronic address:

Interactions of two newly synthesized and six previously reported benzoxanthene lignans (BXLs), analogues of rare natural products, with DNA/RNA, G-quadruplex and HSA were evaluated by a set of spectrophotometric methods. Presence/absence of methoxy and hydroxy groups on the benzoxanthene core and minor modifications at C-1/C-2 side pendants - presence/absence of phenyl ring and presence/absence of methoxy and hydroxy groups on phenyl ring - influenced the fluorescence changes and the binding strength to double-stranded (ds-) and G-quadruplex structures. In general, compounds without phenyl ring showed stronger fluorescence changes upon binding than phenyl-substituted BXLs. On the other hand, BXLs with an unsubstituted phenyl ring showed the best stabilization effects of G-quadruplex. Circular dichroism spectroscopy results suggest mixed binding mode, groove binding and partial intercalation, to ds-DNA/RNA and end-stacking to top or bottom G-tetrads as the main binding modes of BXLs to those targets. All compounds exhibited micromolar binding affinities toward HSA and an increased protein thermal stability. Moderate to strong antiradical scavenging activity was observed for all BXLs with hydroxy groups at C-6, C-9 and C-10 positions of the benzoxanthene core, except for derivative bearing methoxy groups at these positions. BXLs with unsubstituted or low-substituted phenyl ring and one derivative without phenyl ring showed strong growth inhibition of Gram-positive Staphylococcus aureus. All compounds showed moderate to strong tumor cell growth-inhibitory activity and cytotoxicity.
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http://dx.doi.org/10.1016/j.bioorg.2020.104190DOI Listing
November 2020

Mass Spectrometry and H-NMR Study of (Quebracho) Tannins as a Source of Hypoglycemic and Antioxidant Principles.

Molecules 2020 Jul 17;25(14). Epub 2020 Jul 17.

Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

The ethyl acetate extract of the commercial tannin Tan'Activ QS-SOL (from wood), employed for the production of red wine, was subjected to chromatography on Sephadex LH-20, providing nine fractions (A-1-A-9), which were estimated for total phenols content (GAE), antioxidant activity (DPPH, ORAC), and hypoglycemic activity (α-glucosidase and α-amylase inhibition). All the fractions were analyzed by means of HPLC/ESI-MS/MS and H-NMR to identify the principal active constituents. Fractions A-1 and A-3 showed the highest antioxidant activity and gallic acid (), pyrogallol (), eriodictyol (), catechin (), and taxifolin () were identified as the major constituents. The highest α-glucosidase and α-amylase inhibitory activity was observed in fractions A-7-A-9 containing condensed (, , , , , and ) hydrolysable tannins ( and ) as well as esters of quinic acid with different units of gallic acid (, , , , and ). This last class of gallic acid esters are here reported for the first time as α-glucosidase and α-amylase inhibitors.
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http://dx.doi.org/10.3390/molecules25143257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397293PMC
July 2020

Proteomic Analysis of Proteins Responsive to Drought and Low Temperature Stress in a Hard Red Spring Wheat Cultivar.

Molecules 2020 Mar 17;25(6). Epub 2020 Mar 17.

Department of Plant Sciences, University of the Free State, Bloemfontein 9300, South Africa.

Drought stress is becoming more prevalent with global warming, and has been shown tohave large effects on gluten proteins linked to wheat bread making quality. Likewise, lowtemperature stress can detrimentally affect proteins in wheat. This study was done to determine thedifferential abundance of high molecular weight (HMW) glutenin proteins in a drought and lowtemperature stressed high quality hard red spring wheat cultivar (PAN3478), against a control. Thetreatments were applied in the greenhouse at the soft dough stage. HMW glutenin proteins wereextracted from the flour, and were separated by using two-dimensional gel electrophoresis. Proteinspots that had p values lower than 0.05 and fold values equal to or greater than 1.2 were consideredto be significantly differentially abundant. These proteins were further analyzed by using tandemmass spectrometry. There was a 1.3 to 1.8 fold change in 17 protein spots due to the cold treatment.The drought treatment caused a 1.3 to 3.8 fold change in 19 protein spots. These spots matchedeither HMW or low molecular weight (LMW) glutenin subunits. In the latter case, the C subunits ofLMW glutenins were notably found to be up-regulated under both stress conditions. All the proteinsthat have been identified can directly influence dough characteristics. Data are available viaProteomeXchange with the identifier PXD017578.
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http://dx.doi.org/10.3390/molecules25061366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144396PMC
March 2020

Substantial Equivalence of a Transgenic Lemon Fruit Showing Postharvest Fungal Pathogens Resistance.

J Agric Food Chem 2020 Mar 10;68(12):3806-3816. Epub 2020 Mar 10.

Department of Agriculture, Food and Environment, University of Catania, 95123 Catania, Italy.

The development of genetically modified (GM) crops speeds up the obtainment of novel varieties with improved agronomic characteristics. However, the risk evaluation of the use of GMs is mandatory before their release in the market. In this paper, an untargeted and comprehensive nuclear magnetic resonance-based metabolomic study was carried out on the peel and flesh of a transgenic lemon clone (E23) expressing the chit42 gene and exhibiting an increased tolerance to some pathogenic fungi and on its wild type. Results highlighted a substantial equivalence of the metabolomics profile of the transgenic clone compared to the wild type. In addition, an enhanced response of the E23 clone toward fungal pathogens affecting the postharvest management in lemon was evidenced. These results confirm the potential of genetic engineering for the punctual modification of specific agronomic traits without altering the whole pattern of metabolites and open new perspectives for a more sustainable and effective management of specific postharvest diseases in citrus.
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http://dx.doi.org/10.1021/acs.jafc.9b07925DOI Listing
March 2020

Synthesis of Bisphenol Neolignans Inspired by Honokiol as Antiproliferative Agents.

Molecules 2020 Feb 7;25(3). Epub 2020 Feb 7.

Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

Honokiol (2) is a natural bisphenol neolignan showing a variety of biological properties, including antitumor activity. Some studies pointed out 2 as a potential anticancer agent in view of its antiproliferative and pro-apoptotic activity towards tumor cells. As a further contribution to these studies, we report here the synthesis of a small library of bisphenol neolignans inspired by honokiol and the evaluation of their antiproliferative activity. The natural lead was hence subjected to simple chemical modifications to obtain the derivatives 3-9; further neolignans (12a-c, 13a-c, 14a-c, and 15a) were synthesized employing the Suzuki-Miyaura reaction, thus obtaining bisphenols with a substitution pattern different from honokiol. These compounds and the natural lead were subjected to antiproliferative assay towards HCT-116, HT-29, and PC3 tumor cell lines. Six of the neolignans show GI values lower than those of 2 towards all cell lines. Compounds 14a, 14c, and 15a are the most effective antiproliferative agents, with GI in the range of 3.6-19.1 µM, in some cases it is lower than those of the anticancer drug 5-fluorouracil. Flow cytometry experiments performed on these neolignans showed that the inhibition of proliferation is mainly due to an apoptotic process. These results indicate that the structural modification of honokiol may open the way to obtaining antitumor neolignans more potent than the natural lead.
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http://dx.doi.org/10.3390/molecules25030733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037512PMC
February 2020

C-glucosidic ellagitannins and galloylated glucoses as potential functional food ingredients with anti-diabetic properties: a study of α-glucosidase and α-amylase inhibition.

Food Chem 2020 May 28;313:126099. Epub 2019 Dec 28.

Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy. Electronic address:

Diabetes mellitus is a metabolic disorder characterized by hyperglycemia, which can be counteracted by inhibition of α-glucosidase and α-amylase, both involved in the carbohydrate metabolism. Fourteen C-glucosidic ellagitannins and three galloylated glucoses were studied as potential α-glucosidase and α-amylase inhibitors. Most of the compounds were found to be moderate inhibitors of α-amylase, but potent inhibitors of α-glucosidase, showing low-micromolar IC values, far lower than that of the antidiabetic drug acarbose. This selectivity can be an advantage for their possible application as functional food ingredients with anti-diabetic properties because strong α-amylase inhibition generally causes undesired side effects. The best inhibitors were selected for further studies. Intrinsic fluorescence measurements confirmed their high affinity towards α-glucosidase, highlighting a static quenching mechanism. Circular dichroism measurements and kinetics of inhibition indicated that the most active C-glucosidic ellagitannin roburin D (RobD) is a competitive inhibitor, whereas α-pentagalloylglucose (α-PGG) acts as a mixed-type inhibitor.
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http://dx.doi.org/10.1016/j.foodchem.2019.126099DOI Listing
May 2020

Dataset of the metabolic and CM-like protein fractions in old and modern wheat Italian genotypes.

Data Brief 2019 Dec 1;27:104730. Epub 2019 Nov 1.

Laboratory of Organic Mass Spectrometry, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125, Catania, Italy.

The present work reports the first comprehensive proteomic profiling and qualitative comparison of metabolic and Chloroform-Methanol (CM)-like protein fractions extracted from mature kernels of two old Sicilian durum wheat landraces, and , and , an improved durum wheat variety widespread in Italy and other Mediterranean countries and chosen as representative of the most widely commercial cultivars. The data are discussed in the related research article "Qualitative proteomic comparison of metabolic and CM-like protein fractions in old and modern wheat Italian genotypes by a shotgun approach" [1]. The results of this work could be used for investigations to understand the relationship between protein profiles of old and modern wheat genotypes and their potential benefits for human consumption.
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http://dx.doi.org/10.1016/j.dib.2019.104730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859223PMC
December 2019

Qualitative proteomic comparison of metabolic and CM-like protein fractions in old and modern wheat Italian genotypes by a shotgun approach.

J Proteomics 2020 01 16;211:103530. Epub 2019 Oct 16.

Laboratory of Organic Mass Spectrometry, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

The close relationship between diet and health is generally recognized and the growing wellness and consciousness, especially in developed countries, have led to increasing interest for old wheat genotypes, based on perceived health benefits. Although nutritional comparison between old and modern wheat varieties is still controversial, it is generally accepted that old wheat genotypes remained unchanged over the last hundred years. By contrast, modern wheat genotypes are derived by modification of old wheats during the so-called "Green-Revolution" in the second half of the 20th century focusing on obtaining properties in terms of higher grain yield. The present work reports the first comprehensive proteomic profiling and qualitative comparison at the molecular level of metabolic and Chloroform-Methanol (CM)-like protein fractions extracted from mature kernels of two old Sicilian durum wheat landraces, Russello and Timilia Reste Bianche, and Simeto, an improved durum wheat variety widespread in Italy and other Mediterranean countries and chosen as representative of the most widely commercial cultivars. The results obtained reveal that metabolic and CM-like protein fractions of old and modern genotypes present remarkably high similarity with only minor differences. This leads to the conclusion that from a food and nutritional perspective there is a substantial equivalence of the protein composition of the old and modern cultivars. Data are available via ProteomeXchange with identifier PXD014449. BIOLOGICAL SIGNIFICANCE: In recent years consumers have shown growing interest in the old wheat genotypes, which are generally perceived more "natural" and healthier than modern ones. However, comparison of nutritional value for modern and old wheat varieties is still controversial suggesting further studies. In particular proteome analysis of old and modern wheat genotypes is currently ongoing with particular focus on gluten proteins, whereas the metabolic protein fraction has not yet been investigated. In the present study, we conducted a comprehensive proteomic profile and qualitative comparison at the molecular level of metabolic and Chloroform-Methanol (CM)-like protein fractions of the old Sicilian landraces Russello and Timilia Reste Bianche and the modern cultivar Simeto by applying a shotgun approach. The results reveal that the metabolic and CM-like protein fractions of old and modern genotypes are remarkably similar with only minor differences, leading to the conclusion that from a food and nutritional perspective there is a substantial equivalence of these cultivars. These results may contribute to improved understanding of the relationship between protein profiles of old wheat genotypes and their potential benefits for human consumption.
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http://dx.doi.org/10.1016/j.jprot.2019.103530DOI Listing
January 2020

A polyphenol-rich extract from an oenological oak-derived tannin influences in vitro maturation of porcine oocytes.

Theriogenology 2019 Apr 21;129:82-89. Epub 2019 Feb 21.

Dipartimento di Scienze Mediche Veterinarie, Università di Bologna, Bologna, Italy.

Tannins have been demonstrated to have antioxidant and various health benefit properties. The aim of this study was to determine the effect of an ethanol extract (TRE) of a commercial oenological tannin (Quercus robur toasted oak wood, Tan'Activ R) on female gamete using an in vitro model of pig oocyte maturation (IVM) and examining nuclear maturation, cytoplasmic maturation, intracellular GSH and ROS levels and cumulus cell steroidogenesis. To this aim, during IVM performed in medium either supplemented (IVM A) or not supplemented (IVM B) with cysteine and β-mercaptoethanol, TRE was added at different concentrations (0, 1, 5, 10, 20 μg/ml). The addition of TRE at all the concentration tested to either IVM A or IVM B, did not influence oocyte nuclear maturation. When IVM was performed in IVM A, no effect was induced on cytoplasmic maturation by TRE at the concentration of 1, 5 and 10 μg/ml, while TRE 20 μg/ml significantly reduced the penetration rate after IVF (p < 0.05) and the blastocyst rate after parthenogenetic activation (p < 0.01). Oocyte maturation in IVM B, compared to IVM A group, decreased GSH (p < 0.001) and increased ROS (p < 0.01) intracellular levels and in turn impaired oocyte cytoplasmic maturation reducing the ability to sustain male pronuclear formation after IVM (p < 0.001) and the developmental competence after parthenogenetic activation (p < 0.001). TRE supplementation to IVM B significantly reduced ROS production (5, 10, 20 μg/ml TRE) to levels similar to IVM A group, and increased GSH levels (10, 20 μg/ml TRE) compared to IVM B (p < 0.05) without reaching those of IVM A group. TRE supplementation to IVM B at the concentrations of 1, 5 and 10 μg/ml significantly improved (p < 0.001) oocyte cytoplasmic maturation enhancing the ability to sustain male pronuclear formation without reaching, however, IVM A group levels. TRE addition at all the concentration tested to both IVM A and IVM B, did not induce any effect on E2 and P4 secretion by cumulus cells suggesting that the biological effect of the ethanol extract is not exerted thought a modulation of cumulus cell steroidogenesis. In conclusion, TRE, thanks to its antioxidant activity, was partially able to reduce the negative effect of the absence of cysteine and β-mercaptoethanol in IVM B, while TRE at high concentration in IVM A was detrimental for oocyte cytoplasmic maturation underlying the importance of maintaining a balanced redox environment during oocyte maturation.
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http://dx.doi.org/10.1016/j.theriogenology.2019.02.017DOI Listing
April 2019

Synthesis of Rosmarinic Acid Amides as Antioxidative and Hypoglycemic Agents.

J Nat Prod 2019 03 20;82(3):573-582. Epub 2019 Feb 20.

Type 2 diabetes mellitus (T2DM) is an important metabolic disorder for which there is an urgent need for new antidiabetic drugs. α-Glucosidase inhibition is an established protocol for T2DM therapy. Because hyperglycemia causes oxidative tissue damage, the development of agents with both α-glucosidase inhibition and antioxidant activity from natural or natural-derived polyphenols such derivatives of rosmarinic acid (RA) represents an attractive therapeutic option. We report a study on amides 1-10 derived from RA and their evaluation for yeast α-glucosidase inhibition and antioxidant activity (DPPH and ORAC tests). All amides showed higher inhibitory activity than that of RA, were by far more potent than the antidiabetic drug acarbose, and proved to be effective antioxidants. A molecular docking study displayed significant binding interactions of RA amides with the active site of α-glucosidase. This in silico optimization study led to the design and synthesis of amides 9 (IC = 42.3 μM) and 10 (IC = 35.2 μM), showing the most potent α-glucosidase inhibition and good antioxidative properties. A kinetic study showed that 10 acts as a mixed type inhibitor.
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http://dx.doi.org/10.1021/acs.jnatprod.8b01002DOI Listing
March 2019

Bioinspired benzoxanthene lignans as a new class of antimycotic agents: synthesis and spp. growth inhibition.

Nat Prod Res 2020 Jun 13;34(12):1653-1662. Epub 2018 Nov 13.

Department of Chemical Sciences, University of Catania, Catania, Italy.

In this work we synthetized the bioinspired benzoxanthene lignans (BXLs) , , and the phenazine derivative as potential antimycotic agents. MICs and MFCs against strains were determined. In a preliminary screening, compounds , , , , were substantially inactive. Compounds and showed antifungal activity, being able to inhibit the growth of the majority of strains with MIC values in the range 4.6-19.2 µM () and 26.0-104.3 µM (); for three strains, the MICs were lower than those obtained using the antimycotic drug fluconazole. The three BXLs , and showed some MIC values lower than that of fluconazole; was also active against two non strains resistant to fluconazole. Phenazine , although active only against one strain (MIC = 1.3 µM), was one order of magnitude more potent than fluconazole. All the BXLs were fungicidal.
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http://dx.doi.org/10.1080/14786419.2018.1525375DOI Listing
June 2020

A mass spectrometry and H NMR study of hypoglycemic and antioxidant principles from a Castanea sativa tannin employed in oenology.

Food Chem 2018 Dec 23;268:585-593. Epub 2018 Jun 23.

Dipartimento di Scienze Chimiche, Università degli Studi di Catania, V.le A. Doria 6, 95125 Catania, Italy. Electronic address:

The ethanol extract of the commercial tannin Tan'Activ C, (from Castanea sativa wood), employed in oenology, was subjected to chromatography on XAD-16 affording fractions X1-X5, evaluated for total phenols content (GAE), antioxidant activity (DPPH, ORAC), and hypoglycemic activity (α-glucosidase inhibition). Fraction X3 showed GAE, radical scavenging activity, and α-glucosidase inhibition higher than those of the Castanea sativa extract, and was subjected to chromatography on Sephadex LH-20 to obtain fractions S1-S7, analyzed by HPLC/ESI-MS/MS and H NMR to identify the main active constituents. In fractions with higher antioxidant activity, gallic acid (4), grandinin (5), valoneic acid dilactone (9), 1,2,3-tri-O-galloyl-β-glucose (14), 3,4,6-tri-O-galloyl-β-glucose (15) and galloyl derivative of valoneic acid dilactone (21) were identified as the major constituents. The highest hypoglycemic activity was found in fractions S6 and especially S7; the major constituents of these fractions are valoneic acid dilactone (9), three tetragalloyl glucose isomers (16-18) and 1,2,3,4,6-penta-O-galloyl-β-glucose (23), previously reported as α-glucosidase inhibitors.
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http://dx.doi.org/10.1016/j.foodchem.2018.06.117DOI Listing
December 2018

Identification by Inverse Virtual Screening of magnolol-based scaffold as new tankyrase-2 inhibitors.

Bioorg Med Chem 2018 08 20;26(14):3953-3957. Epub 2018 Jun 20.

Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Italy. Electronic address:

The natural product magnolol (1) and a selection of its bioinspired derivatives 2-5, were investigated by Inverse Virtual Screening in order to identify putative biological targets from a panel of 308 proteins involved in cancer processes. By this in silico analysis we selected tankyrase-2 (TNKS2), casein kinase 2 (CK2) and bromodomain 9 (Brd9) as potential targets for experimental evaluations. The Surface Plasmon Resonance assay revealed that 3-5 present a good affinity for tankyrase-2, and, in particular, 3 showed an antiproliferative activity on A549 cells higher than the well-known tankyrase-2 inhibitor XAV939 used as reference compound.
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http://dx.doi.org/10.1016/j.bmc.2018.06.019DOI Listing
August 2018

Expanded Newborn Screening Using Tandem Mass Spectrometry: Seven Years of Experience in Eastern Sicily.

Int J Neonatal Screen 2018 Jun 5;4(2):12. Epub 2018 Apr 5.

Referral Center for Inherited Metabolic Diseases, Pediatric Clinical, AOU Policlinico-VE, Via Santa Sofia 78, 95123 Catania, Italy.

The expanded newborn screening for selected inborn errors of metabolism (IEM) in Sicily was introduced in 2007 by a Regional project entitled "Early detection of congenital metabolic diseases: expanded neonatal screening". It established two newborn screening laboratories, for Western and Eastern Sicily, which started their activity in 2011. Here we present the results of expanded screening (excluding phenylketonuria (PKU)) of the Eastern laboratory from January 2011 to December 2017. Our data highlight the importance of the expanded newborn screening as a basic health program to avoid the underestimation of rare diseases and the need of further investigations even when there are no textbook alterations of the metabolic profiles. We performed our analysis on dried blood spot by tandem mass spectrometry, according to Italian guidelines. A total of 196 samples from 60,408 newborns gave positive screening results (recall rate 0.32%) while 12 babies were true positive, including 2 newborns whose mothers resulted in being affected by a metabolic disease. The overall frequency of IEM found in the screening panel was 1:6041 (mothers excluded) or 1:5034 (mothers included). The introduction of MS/MS technology in Sicily has significantly increased the detection of inherited metabolic disorders, including those not previously covered, with a predictable improved outcome for several disorders.
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http://dx.doi.org/10.3390/ijns4020012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510204PMC
June 2018

Polymorphism at donkey β-lactoglobulin II locus: identification and characterization of a new genetic variant with a very low expression.

Amino Acids 2018 Jun 23;50(6):735-746. Epub 2018 Mar 23.

Animal Production Section, Department of Agriculture, Food and Environment, University of Catania, Via Valdisavoia 5, 95123, Catania, Italy.

In the last years, donkey milk had evidenced a renewed interest as a potential functional food and a breast milk substitute. In this light, the study of the protein composition assumes an important role. In particular, β-lactoglobulin (β-LG), which is considered as one of the main allergenic milk protein, in donkey species consists of two molecular forms, namely β-LG I and β-LG II. In the present research, a genetic analysis coupled with a proteomic approach showed the presence of a new allele, here named F, which is apparently associated with a null or a severely reduced expression of β-LG II protein. The new β-LG II F genetic variant shows a theoretical average mass (M) of 18,310.64 Da, a value practically corresponding with that of the variant D (∆ < 0.07 Da), but differs from β-LG II D for two amino acid substitutions: Thr (variant F) → Ala (variant D) and Thr (variant F) → Met (variant D). Proteomic investigation of the whey protein fraction of an individual milk sample, homozygous FF at β-LG II locus, allowed to identify, as very minor component, the new β-LG II F genetic variant. By MS/MS analysis of enzymatic digests, the sequence of the β-LG II F was characterized, and the predicted genomic data confirmed.
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http://dx.doi.org/10.1007/s00726-018-2555-1DOI Listing
June 2018

Biological effects of polyphenol-rich extract and fractions from an oenological oak-derived tannin on in vitro swine sperm capacitation and fertilizing ability.

Theriogenology 2018 Mar 14;108:284-290. Epub 2017 Dec 14.

Dipartimento di Scienze Mediche Veterinarie, Università di Bologna, Bologna, Italy.

Although excessive ROS levels induce sperm damage, sperm capacitation is an oxidative event that requires low amounts of ROS. As the antioxidant activity of the ethanol extract (TRE) of a commercial oenological tannin (Quercus robur toasted oak wood, Tan'Activ R) and its four fractions (FA, FB, FC, FD) has been recently reported, the present study was set up to investigate the biological effects of TRE and its fractions in an in vitro model of sperm capacitation and fertilization. Boar sperm capacitation or gamete coincubation were performed in presence of TRE or its fractions (0, 1, 10, 100 μg/ml). TRE at the concentration of 10 μg/ml (TRE10) stimulated sperm capacitation, as it increased (p < .001) the percentage of spermatozoa with tyrosine-phosphorylated protein positivity in the tail principal piece (B pattern) (67.0 ± 10.6 vs. 48.6 ± 9.0, mean ± SD for TRE10 vs. Ctr respectively). Moreover T10 significantly (p < .001) increased oocyte fertilization rate (91.9 ± 4.0 vs. 69.0 ± 14.8, TRE10 vs. Ctr respectively). An opposite effect of TRE at the concentration of 100 μg/ml (TRE100) on both sperm capacitation (B pattern cell percentage 33.3 ± 29.2) and fertilizing ability (fertilization rate 4.9 ± 8.3), associated with a higher sperm viability (66.9 ± 9.3 vs. 35.4 ± 10.8, TRE100 vs. Ctr respectively) (p < .001), was recorded. The potency of the TRE fractions seems to be highest in FB followed by FC, faint in FD and nearly absent in FA. Our results show that TRE and its fractions, in a different extent, exert a powerful biological effect in finely modulating capacitation and sperm fertilizing ability.
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http://dx.doi.org/10.1016/j.theriogenology.2017.12.015DOI Listing
March 2018

Proteins and bioactive peptides from donkey milk: The molecular basis for its reduced allergenic properties.

Food Res Int 2017 09 4;99(Pt 1):41-57. Epub 2017 Jul 4.

Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

The legendary therapeutics properties of donkey milk have recently been supported by many clinical trials who have clearly demonstrated that, even if with adequate lipid integration, it may represent a valid natural substitute of cow milk for feeding allergic children. During the last decade many investigations by MS-based methods have been performed in order to obtain a better knowledge of donkey milk proteins. The knowledge about the primary structure of donkey milk proteins now may provide the basis for a more accurate comprehension of its potential benefits for human nutrition. In this aspect, experimental data today available clearly demonstrate that donkey milk proteins (especially casein components) are more closely related with the human homologues rather than cow counterparts. Moreover, the low allergenic properties of donkey milk with respect to cow one seem to be related to the low total protein content, the low ratio of caseins to whey fraction, and finally to the presence in almost all bovine IgE-binding linear epitopes of multiple amino acid differences with respect to the corresponding regions of donkey milk counterparts.
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http://dx.doi.org/10.1016/j.foodres.2017.07.002DOI Listing
September 2017

Chain-breaking antioxidant activity of hydroxylated and methoxylated magnolol derivatives: the role of H-bonds.

Org Biomol Chem 2017 Jul;15(29):6177-6184

Department of Chemical Sciences, University of Catania, Viale A. Doria 6, I-95125 Catania, Italy.

Chemical modification of magnolol, an uncommon dimeric neolignan contained in Magnolia genus trees, provides a unique array of polyphenols having interesting biological activity potentially related to radical scavenging. The chain-breaking antioxidant activity of four new hydroxylated and methoxylated magnolol derivatives was explored by experimental and computational methods. The measurement of the rate constant of the reaction with ROO˙ radicals (k) in an apolar solvent showed that the introduction of hydroxyl groups ortho to the phenolic OH in magnolol increased the k value, being 2.4 × 10 M s and 3.3 × 10 M s for the mono and the dihydroxy derivatives respectively (k of magnolol is 6.1 × 10 M s). The di-methoxylated derivative is less reactive than magnolol (k = 1.1 × 10 M s), while the insertion of both hydroxyl and methoxyl groups showed no effect (6.0 × 10 M s). Infrared spectroscopy and theoretical calculations allowed a rationalization of these results and pointed out the crucial role of intramolecular H-bonds. We also show that a correct estimation of the rate constant of the reaction with ROO˙ radicals, by using BDE(OH) calculations, requires that the geometry of the radical is as close as possible to that of the parent phenol.
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http://dx.doi.org/10.1039/c7ob01195dDOI Listing
July 2017

Comparative proteomic analysis of two transgenic low-gliadin wheat lines and non-transgenic wheat control.

J Proteomics 2017 08 15;165:102-112. Epub 2017 Jun 15.

Department of Plant Breeding, Institute for Sustainable Agriculture (IAS-CSIC), 14004 Córdoba, Spain. Electronic address:

Gluten proteins are major determinants of the bread making quality of wheat, but also of important wheat-related disorders, including coeliac disease (CD), and allergies. We carried out a proteomic study using the total grain proteins from two low-gliadin wheat lines, obtained by RNAi, and the untransformed wild type as reference. The impact of silencing on both target and on non-target proteins was evaluated. Because of the great protein complexity, we performed separate analyses of four kernel protein fractions: gliadins and glutenin subunits, and metabolic and CM-like proteins, by using a classical 2D electrophoresis gel based approach followed by RP-HPLC/nESI-MS/MS. As a result of the strong down-regulation of gliadins, the HMW-GS, metabolic and chloroform/methanol soluble proteins were over-accumulated in the transgenic lines, especially in the line D793, which showed the highest silencing of gliadins. Basing on these data, and considering that metabolic proteins and chloroform/methanol soluble proteins (CM-like), such as the α-amylase/trypsin inhibitor family, β-amylase and serpins, were related to wheat allergens, further in vivo analysis will be needed, especially those related to clinical trials in controlled patients, to determine if these lines could be used for food preparation for celiac or other gluten intolerant groups.

Biological Significance: Several enteropathies and allergies are related to wheat proteins. Biotechnological techniques such as genetic transformation and RNA interference have allowed the silencing of gliadin genes, providing lines with very low gliadin content in the grains. We report a proteomic-based approach to characterize two low-gliadin transgenic wheat lines obtained by RNAi technology. These lines harbor the same silencing fragment, but driven by two different endosperm specific promoters (γ-gliadin and D-hordein). The comprehensive proteome analysis of these transgenic lines, by combining two-dimensional electrophoresis and RP-HPLC/nESI-MS/MS, provided a large number of spots differentially expressed between the control and the transgenic lines. Hence, the results of this study will facilitate further safety evaluation of these transgenic lines.
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http://dx.doi.org/10.1016/j.jprot.2017.06.010DOI Listing
August 2017

Chemoenzymatic Synthesis and α-Glucosidase Inhibitory Activity of Dimeric Neolignans Inspired by Magnolol.

J Nat Prod 2017 05 12;80(5):1648-1657. Epub 2017 May 12.

Dipartimento di Scienze Chimiche, Università degli Studi di Catania , Viale A. Doria 6, I-95125 Catania, Italy.

A chemoenzymatic synthesis of a small library of dimeric neolignans inspired by magnolol (1) is reported. The 2-iodoxybenzoic acid (IBX)-mediated regioselective ortho-hydroxylation of magnolol is described, affording the bisphenols 6 and 7. Further magnolol analogues (12, 13, 15-17, 19-23) were obtained from eugenol (3), tyrosol (4), and homovanillic alcohol (5), through horseradish peroxidase (HRP)-mediated oxidative coupling and regioselective ortho-hydroxylation or ortho-demethylation in the presence of IBX, followed by reductive treatment with NaSO. A chemoselective protection/deprotection of the alcoholic group of 4 and 5 was carried out by lipase-mediated acetylation/deacetylation. The dimeric neolignans, together with 1 and honokiol (2), were evaluated as inhibitors of yeast α-glucosidase, in view of their possible utilization and optimization as antidiabetic drugs. The synthetic analogues of magnolol showed a strong inhibitory activity with IC values in the range 0.15-4.1 μM, much lower than those of honokiol and the reference compounds quercetin and acarbose. In particular, a very potent inhibitory activity, with an IC of 0.15 μM, was observed for 1,1'-dityrosol-8,8'-diacetate (15), and comparable inhibitory activities were also shown by bisphenols 6 (0.49 μM), 13 (0.50 μM), and 22 (0.86 μM). A kinetic study showed that 15 acts as a competitive inhibitor, with a K value of 0.86 μM.
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http://dx.doi.org/10.1021/acs.jnatprod.7b00250DOI Listing
May 2017

Polyphemus, Odysseus and the ovine milk proteome.

J Proteomics 2017 01 23;152:58-74. Epub 2016 Oct 23.

Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

In the last years the amount of ovine milk production, mainly used to formulate a wide range of different and exclusive dairy products often categorized as gourmet food, has been progressively increasing. Taking also into account that sheep milk (SM) also appears to be potentially less allergenic than cow's one, an in-depth information about its protein composition is essential to improve the comprehension of its potential benefits for human consumption. The present work reports the results of an in-depth characterization of SM whey proteome, carried out by coupling the CPLL technology with SDS-PAGE and high resolution UPLC-nESI MS/MS analysis. This approach allowed the identification of 718 different protein components, 644 of which are from unique genes. Particularly, this identification has expanded literature data about sheep whey proteome by 193 novel proteins previously undetected, many of which are involved in the defence/immunity mechanisms or in the nutrient delivery system. A comparative analysis of SM proteome known to date with cow's milk proteome, evidenced that while about 29% of SM proteins are also present in CM, 71% of the identified components appear to be unique of SM proteome and include a heterogeneous group of components which seem to have health-promoting benefits. The data have been deposited to the ProteomeXchange with identifier .
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http://dx.doi.org/10.1016/j.jprot.2016.10.007DOI Listing
January 2017

The starch-bound alpha-amylase/trypsin-inhibitors in Avena.

Mol Genet Genomics 2016 Dec 5;291(6):2043-2054. Epub 2016 Aug 5.

Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria (CREA-QCE), Via Manziana 30, 00189, Rome, Italy.

Oat kernels exhibit an extra-soft texture, a trait recently demonstrated to be largely modulated by starch-bound tryptophan-rich 2S proteins, the vromindolines. In this study, fractionation by two-dimensional electrophoresis of starch-bound proteins in 25 oat (Avena sativa) cultivars and 11 diploid or tetraploid Avena species revealed novel 2S proteins called Avena α-amylase/trypsin-inhibitors (AATI) because of their sequence similarity with wheat α-amylase/trypsin inhibitors. Thirty-seven AATI polypeptides, about 14 kDa in size, were split into three families named AATI-1, AATI-2, and AATI-3 with different primary structures and isoelectric points. AATI-1 and AATI-2 proteins showed 55.5-60.0 % sequence similarity with wheat α-amylase inhibitors CM1, CM2, and CM16, which have been found to cause innate immunity responses in celiac disease and non-celiac gluten sensitivity. Diploid A-genome and tetraploid AC-genome oat species possess three and five genes encoding for the AATI proteins, respectively, whereas hexaploid A. sativa exhibits 12 genes dispersed over the A-, C-, and D-genomes. Some AATI proteins expressed in hexaploid oats were assigned to the A-genome based on similarity to their counterparts in diploid species, contributing to further clarify the genetic origin of hexaploid oats. Moreover, AATI may interact with starch-bound vromindolines in determining the extra-soft texture of oat kernels and, due to their balanced amino acid compositions, may contribute to the biological value of oat proteins in a positive manner.
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http://dx.doi.org/10.1007/s00438-016-1238-4DOI Listing
December 2016

Site-specific glycosylation of donkey milk lactoferrin investigated by high-resolution mass spectrometry.

Amino Acids 2016 12 22;48(12):2799-2808. Epub 2016 Aug 22.

Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark.

A comprehensive monosaccharide composition of the N-glycans of donkey milk lactoferrin, isolated by ion exchange chromatography from an individual milk sample, was obtained by means of chymotryptic digestion, TiO and HILIC enrichment, reversed-phase high-performance liquid chromatography, electrospray mass spectrometry, and high collision dissociation fragmentation. The results obtained allowed identifying 26 different glycan structures, including high mannose, complex and hybrid N-glycans, linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476. Altogether, the N-glycan structures determined revealed that most of the N-glycans identified in donkey milk lactoferrin are neutral complex/hybrid. Indeed, 10 neutral non-fucosylated complex/hybrid N-glycans and 4 neutral fucosylated complex/hybrid N-glycans were found. In addition, two high mannose N-glycans, four sialylated fucosylated complex N-glycans and six sialylated non-fucosylated complex N-glycans, one of which containing N-glycolylneuraminic acid (Neu5Gc), were found. A comparison of the monosaccharide composition of the N-glycans of donkey milk lactoferrin with respect to that of human, bovine and goat milk lactoferrin is reported. Data are available via ProteomeXchange with identifier PXD004289.
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http://dx.doi.org/10.1007/s00726-016-2315-zDOI Listing
December 2016

α-Glucosidase inhibition and antioxidant activity of an oenological commercial tannin. Extraction, fractionation and analysis by HPLC/ESI-MS/MS and (1)H NMR.

Food Chem 2017 Jan 25;215:50-60. Epub 2016 Jul 25.

Dipartimento di Scienze Chimiche, Università degli Studi di Catania, V.le A. Doria 6, 95125 Catania, Italy. Electronic address:

Two batches of the oenological tannin Tan'Activ R, (toasted oak wood - Quercus robur), were extracted with ethanol. A fractionation on XAD-16 afforded four fractions for each extract. Extracts and fractions were evaluated for antioxidant activity (DPPH), polyphenol content (GAE) and yeast α-glucosidase inhibitory activity. Comparable results were obtained for both columns, fractions X1B and X2B showing the highest antioxidant activity. Fractions X1C and X2C notably inhibited α-glucosidase, with IC50=9.89 and 8.05μg/mL, respectively. Fractions were subjected to HPLC/ESI-MS/MS and (1)H NMR analysis. The main phenolic constituents of both X1B and X2B were a monogalloylglucose isomer (1), a HHDP-glucose isomer (2), castalin (3) gallic acid (4), vescalagin (5), and grandinin (or its isomer roburin E, 6). X1C and X2C showed a complex composition, including non-phenolic constituents. Fractionation of X2C gave a subfraction, with enhanced α-glucosidase inhibitory activity (IC50=6.15μg/mL), with castalagin (7) as the main constituent.
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http://dx.doi.org/10.1016/j.foodchem.2016.07.136DOI Listing
January 2017

Sequence characterization and glycosylation sites identification of donkey milk lactoferrin by multiple enzyme digestions and mass spectrometry.

Amino Acids 2016 07 28;48(7):1569-80. Epub 2016 Mar 28.

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense, Denmark.

Lactoferrin, a protein showing an array of biochemical properties, including immuno-modulation, iron-binding ability, as well as antioxidant, antibacterial and antiviral activities, but which may also represent a potential milk allergen, was isolated from donkey milk by ion exchange chromatography. The characterization of its primary structure, by means of enzymatic digestions, SPITC derivatization of tryptic digest, reversed-phase high performance liquid chromatography, electrospray and matrix-assisted laser desorption/ionization mass spectrometry, is reported. Our results allowed the almost complete characterization of donkey lactoferrin sequence, that, at least for the covered sequence, differs from the horse genomic deduced sequence (UniProtKB Acc. Nr. O77811) by five point substitutions located at positions 91 (Arg → His), 328 (Thr → Ile/Leu), 466 (Ala → Gly), 642 (Asn → Ser) and 668 (Ser → Ala). Analysis of the glycosylated protein showed that glycans in donkey lactoferrin are linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476.
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http://dx.doi.org/10.1007/s00726-016-2209-0DOI Listing
July 2016

Zeus, Aesculapius, Amalthea and the proteome of goat milk.

J Proteomics 2015 Oct 17;128:69-82. Epub 2015 Jul 17.

Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

The goat whey proteome has been explored in depth via capture with combinatorial peptide ligand libraries (CPLLs) at three different pH values. A total of 452 unique species have been tabulated, a proteome discovery so far unmatched in any single other investigation of milk from any mammalian species. This massive discovery is probably related to: i) the extraordinary load of proteins onto the CPLL beads (i.e. 2 g for each different pH captures) vs. barely 100 μL of beads; ii) the high resolution/high mass accuracy of mass spectral data; and iii) the use of two complementary tools, Mascot and PEAKS, each one contributing to a set of unique protein IDs. Due to the relative paucity of available protein annotations for goat, only 10% of the identified proteins belong to the capra, whereas 52% are specific of sheep and 37% are homologous to that of bovine milk. This work reports the largest description so far of the goat milk proteome, which has been compared with cow's milk proteome and would thus help to understand the importance of low-abundance proteins with respect to the unique biological properties of this nutrient.
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http://dx.doi.org/10.1016/j.jprot.2015.07.009DOI Listing
October 2015

Protein profile of exhaled breath condensate determined by high resolution mass spectrometry.

J Pharm Biomed Anal 2015 Feb 5;105:134-149. Epub 2014 Dec 5.

Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

A method based on liquid chromatography/high resolution tandem mass spectrometry coupled with electrophoretic separation, for determination and relative quantification of the protein composition of exhaled breath condensate (EBC), was developed. Application of the procedure to a sample of EBC, pooled from nine healthy subjects, resulted in the identification of 167 unique gene products, 113 of which not previously reported in EBC samples. The abundance of the protein identified was estimated by means of the exponentially modified protein abundance index protocol (emPAI). Cytokeratins were by far the most abundant proteins in EBC samples. Many of the identified proteins were associated with multiple cellular location with cytoplasm constituting the largest group. Cytosol, nucleus, membrane, cytoskeleton and extracellular were other abundantly represented locations. No amylase was detected, suggesting the absence of saliva protein contamination. The profile obtained represents the most comprehensive protein characterization of EBC so far reported and demonstrates that this approach provides a powerful tool for investigating the protein profile of EBC samples. Compared with analogous investigations, this study also shows that the protein profile of EBC is strongly affected by the sampling method adopted.
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http://dx.doi.org/10.1016/j.jpba.2014.11.050DOI Listing
February 2015