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Italy is one of the countries on track with the WHO's agenda to eliminate hepatitis C virus (HCV) by 2030. Healthcare facilities play a crucial role in seeking patients who are infected but have not yet been treated. We assessed the effectiveness of a recall strategy, named 'Telepass' project, for patients exposed to HCV infection who have not yet been linked to care in a large tertiary care centre. The 'Telepass' project was structured in two phases: (a) a retrospective analysis first identified all anti-HCV-positive subjects among patients who underwent pre-operative assessment in the facility in the course of one year; (b) a following prospective phase, aimed to recall patients in need either of further diagnostic tests (ie HCV-RNA) or treatment. A total of 12246 records of patients tested for HCV antibodies were reviewed. The overall prevalence of anti-HCV-positive subjects was 1.83% (224/12246) with a male/female ratio of 2.07. Out of the 224 anti-HCV-positive patients, 123 (54.91%) did not have documented HCV-RNA tests and were therefore selected for recall. Of these, 123 were reachable and 26 (21.13%) were successfully linked to care. Ten patients (38.46%) tested HCV-RNA positive and initiated treatment with direct-acting antivirals (DAAs). The Telepass study highlights that a recall strategy starting from internal hospital databases can help identify patients with chronic HCV infection who have not yet been linked to care, and provides an epidemiological insight into the prevalence of HCV infection in Italy in the late DAAs era., (© 2021 John Wiley & Sons Ltd.)

The advanced glycation end-products (AGEs) arise from non-enzymatic reactions of sugar with protein side chains, some of which are oxido-reductive in nature. Enhanced production of AGEs plays an important role in the pathogenesis of diabetic complications as well as in natural aging, renal failure, oxidative stress, and chronic inflammation. The aim of this work is to study antiglycation effects of polyphenol compounds extracted by hazelnut skin that represents an example of polyphenols-rich food industry by-product, on AGEs formation. AGEs derived from incubation of bovine serum albumin (BSA) and methylglyoxal (MGO) were characterized by fluorescence. The phenolics identification and total polyphenol content in hazelnut skin extracts were analyzed by HPLC-MS and the Folin-Ciocalteu method, respectively. Antioxidant efficacy was evaluated by monitoring total antioxidant activity to assess the ABTS radical scavenging activity of samples by TEAC assay and oxygen radical absorbance capacity (ORAC) assay, expressed as millimoles of Trolox equivalents per gram of sample. Data here presented suggest that phenolic compounds in hazelnut skin have an inhibitory effect on the BSA-AGEs model in vitro, and this effect is concentration-dependent. The putative role of the hazelnut skin antioxidative properties for hindering AGEs formation is also discussed. Because of AGEs contribution to the pathogenesis of several chronic diseases, foods enriched, or supplements containing natural bioactive molecules able to inhibit their production could be an interesting new strategy for supporting therapeutic approaches with a positive effect on human health.

A HPLC-DAD/ESI-MS method has been developed and validated for the analysis of the most representative phenolic compounds in extra-virgin olive oil (EVOO) samples using a green extraction approach based on deep eutectic solvents (DESs) at room temperature. We examined ten DESs based on choline chloride and betaine in combination with different hydrogen bond donors comprising six alcohols, two organic acids, and one urea. Five phenolic compounds, belonging to the classes of secoiridoids and phenolic alcohols, were selected for the evaluation of extraction efficiency. A betaine-based DES with glycerol (molar ratio 1:2) was found to be the most effective for extracting phenolic compounds as compared to a conventional solvent. The optimization of the extraction method involved the study of the quantity of water to be added to the DES and evaluation of the sample-to-solvent ratio optimal condition. Thirty percent of water added to DES and sample to solvent ratio 1:1 (w/v) were selected as the best conditions. The chromatographic method was validated by studying LOD, LOQ, intraday and interday retention time precision, and linearity range. Recovery values obtained spiking seed oil sample aliquots with standard compounds at 5 and 100 μg/g concentration were in the range between 75.2% and 98.7%., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Coffee beans contain several compounds which have shown nutraceutial properties; among them chlorogenic acids (CGAs) are the most studied, have shown nutraceutical properties. These molecules are also present in the waste by-product known as spent coffee grounds (SCG). SCG are generated during the production of coffee drink, whereby roasted, ground coffee beans are heat or steam treated. A HPLC-PDA/ESI-MS method has been developed for the analysis of the most representative CGAs in SCG samples using a green extraction approach based on deep eutectic solvents (DESs). We examined fifteen different DESs based on choline chloride and betaine as hydrogen bond acceptor in combination with different hydrogen bond donors being seven alcohols, two organic acids, one sugar and urea. Fifteen CGAs were detected, identified and quantified for the evaluation of extraction efficiency. A betaine-based DES with triethylene glycol (molar ratio 1:2) was the most effective one at extracting CGAs from SCG also compared to a conventional organic solvent. Thirty percent of water added to DES and sample to solvent ratio 1:15 (g/mL) were selected as the best extraction conditions. The chromatographic method was validated studying LOD, LOQ, retention time and peak area precision, and linearity range. Retention time and peak area repeatability were determined obtaining RSDs values below 0.63 % and 8.51 %, respectively. Inter-day precision was determined obtaining RSDs value below 0.58 % and 8.80 % for retention time and peak area, respectively., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In the present study separation of enantiomers of some chiral neutral, basic and weakly acidic analytes was investigated on the chiral stationary phase (CSP) made by covalent immobilization of amylose tris(3-chloro-5-methylphenylcarbamate) onto aminopropylsilanized (APS) silica in nano-liquid chromatography (nano-LC) in aqueous methanol or acetonitrile mixtures. It has been shown that similar to high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) this chiral selector is useful for separation of enantiomers of neutral, basic and acidic analytes also in nano-LC. In comparison to our previous research, in which the chiral selector (CS) was bonded on native silica, in this study, the CS was immobilized on APS silica in order to improve chromatographic performance towards basic analytes. In fact, some improvement was observed and surprisingly not only for basic but also for neutral and acidic analytes. Again, quite unexpectedly almost no electroosmotic flow (EOF) was observed in capillaries packed with ca. 20% (w/w) amylose tris(3-chloro-5-methylphenylcarbamate) immobilized onto APS silica although the same APS silica before attachment of chiral selector exhibited significant EOF. In order to generate EOF in the capillaries with the CSP and enable capillary electrochromatographic (CEC) experiment on it, the short segment of the capillary column was packed with APS silica without chiral selector. The EOF in such capillary enabled CEC experiment and some preliminary results are reported here., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In the present study separation of enantiomers of some chiral neutral and weakly acidic analytes was investigated on the chiral stationary phase (CSP) made by covalent immobilization of amylose tris(3-chloro-5-methylphenylcarbamate) onto silica in nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC) in acetonitrile and aqueous acetonitrile. Few comparisons were made also between the enantioseparations in nano-LC and high-performance liquid chromatography (HPLC) with the chiral column of 4.6 × 250 mm dimension. Slightly better separation of enantiomers was observed in HPLC mode compared to nano-LC mode. It was shown that in the capillary columns packed with the CSP containing about 20% (w/w) of a covalently immobilized neutral chiral selector, amylose tris(3-chloro-5-methylphenylcarbamate), sufficient electroosmotic flow has been generated and enantioseparations with reasonable analysis time were performed also in CEC mode. It was shown once again that CEC offers a clear advantage over nano-LC from the viewpoint of plate numbers and peak resolution., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The chiral separation of baclofen (Bac) was obtained by nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) using a 100 μm I.D. fused silica capillary column packed with silica particles chemically modified with vancomycin. Various experimental parameters, such as composition (buffer concentration, water content, organic modifier) and pH of the mobile phase and sample solvent were investigated for method optimization. In order to increase the sensitivity an on-column focusing procedure was applied. Acceptable separation of Bac enantiomers was obtained in less than 11 min eluting in isocratic mode, with 90:10 MeOH/water (v/v) containing 10 mM ammonium acetate at pH 4.5. These optimized experimental conditions were applied to the analysis of human plasma samples spiked with racemic mixture of Bac. The use of a Buckypaper disc as sorbent membrane allows one to recover both enantiomers with yields ≥ 65%. The method was fully validated, following the identification criteria of the European Commission Decision 2002/657/EC., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This paper illustrates the development of a procedure based on the use of a low transition temperature mixture (LTTM) for the dispersive liquid-liquid microextraction (DLLME) of fungicides, insecticides and acaricides from surface waters. The LTTM preparation involves the heat-mixing of choline chloride and acetylsalicylic acid in a molar ratio 1:2 (ChCl(ASA) 2 ). The resulting mixture appears as a clear viscous liquid at room-temperature, denser than water (1.20 ± 0.01 g mL -1 ). For its characterization, differential scanning calorimetry (DSC) provided crucial evidence to classify it as a LTTM rather than as a deep eutectic solvent (DES) since it revealed an intense glass transition at -37 °C. Large-angle X-ray scattering (LAXS) confirmed the lack of any long-distance order. Due to the LTTM immiscibility with water, an evaluation study was carried out to test ChCl(ASA) 2 as an effective alternative to the conventional chlorinated solvents for DLLME. To this end, 24 pesticides were used as model compounds, extracted from surface water samples (5 mL) and analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The definitive procedure required the optimization of some key parameters such as volume of extracting solvent, type and volume of dispersing solvent, volume of the aqueous sample, LTTM dispersion procedure, and extraction time. Depending on pKa and logP values, recoveries ranged from 18 (for very polar compounds) to 96%, revealing that the ideal candidates for the extraction with ChCl(ASA) 2 are neutral compounds with logP >2. After complete validation, the method was applied to analyze water samples from the River Tiber where dodine and dimetomorph were found at low μg L -1 concentration levels., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This work deals with the potentiality of nano liquid chromatography (Nano-LC) for the chiral separation of racemic mixture of tryptophan and some selected derivatives by using 100 µm i.d. fused silica capillary packed with teicoplanin bonded to 5 µm diol silica stationary phase. The experiments were carried out by using a cheap and laboratory-assembled nano-LC-UV system. Elution was done in an isocratic mode using a polar organic mobile phase. In order to find the optimum chiral separation of the studied enantiomers, some chromatographic experimental parameters were systematically studied and optimized. Among them, mobile phase composition, namely organic modifier type and concentration, buffer type and pH and aqueous content and sample solvent dilution on retention time, retention factor and enantioresolution factor were studied. Baseline enantioresolution and good peak shape was achieved utilizing the mobile phase containing 40 mM ammonium formate at pH pH 2.5 in ACN/water/acetone (60:30:10, v/v/v) at 520 nL/min in less than 8 min analysis time., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The technologically interesting properties of palladium nanoparticles (Pd-NPs) allowed their widespread industrial application, although concerns emerged on increasing general and occupational levels of exposure. In this context, to assess the toxicological behavior of Pd-NPs, and particularly their endocrine disruptive potential, has become a public health priority. Therefore, we evaluated Pd-NP impact on the female endocrine reproductive system of Wistar rats sub-chronically treated for 90 days with increasing doses of this xenobiotic (0.12, 1.2, and 12 µg/kg, administered at days 1, 30, and 60 for cumulative doses of 0.36, 3.6, and 36 µg/kg) via the intravenous route. In this regard, we investigated potential alterations in different sex hormone, for example, estradiol, follicle-stimulating hormone (FSH), luteinizing hormone, progesterone, and testosterone, serum concentrations. All treated groups showed significantly greater levels of FSH compared to controls, suggesting a possible impact of Pd-NPs on the regulatory system that controls the normal physiology of female reproductive function. Although relevant, since obtained under sub-chronic, low-dose conditions of exposure resembling those encountered in real-world scenarios, the present results are preliminary and require confirmation as well as identification of the possible underlining molecular mechanisms. From a public and occupational health perspective, implications for the reproductive health of exposed subjects and the next generations of women exposed during their childbearing age or pregnancy should be elucidated. This information is essential to elaborate adequate preventive strategies for assessing and controlling possible Pd-NPs adverse effects on the endocrine system.

The recently developed therapeutic strategies have led to unprecedented improvements in the control of metastatic melanoma and in the survival of specific subgroups of patients. However, drug resistance, low response rates, and undesired side effects make these treatments not suitable or tolerable for all the patients, and chemotherapeutic treatments appear still indispensable, at least for subgroups of patients. New combinatory strategies are also under investigation as tailored treatments or salvage therapies, including combined treatments of immunotherapy with conventional chemotherapy. On this basis, and in consideration of the antineoplastic properties of ω-3 polyunsaturated fatty acids, we have here investigated the potential of these bioactive dietary factors to revert the resistance frequently exhibited by this form of cancer to cisplatin (CDDP, cis-diamminedichloroplatinum). We demonstrated that docosahexenoic acid (DHA, 22:6ω-3) sensitizes the cells to the CDDP-induced inhibition of cell growth and migration by reverting CDDP effects on DNA damage and ERCC1 expression, as well as on the DUSP6 and p-ERK expressions, which regulate ERCC1 activation upwardly. In line, DUSP6 gene silencing prevented the effect of DHA, confirming that DHA acted on the DUSP6/p-ERK/ERCC1 repair pathways to sensitize melanoma cells to the anticancer effect of CDDP. Similar effects were obtained also with eicosapentaenoic acid (20:5ω-3). Overall, our findings suggest that the combination of CDDP treatment with a dietary supplementation with ω-3 polyunsaturated fatty acids could potentially represent a new therapeutic strategy for overcoming CDDP resistance in metastatic melanoma.

Patients with non-celiac gluten sensitivity (NCGS) do not have celiac disease, but their symptoms improve after a gluten-free diet (GFD). However, to date, it is uncertain if gluten or other components of wheat are responsible for these symptoms. The aim of this study was to compare the effects of an organic durum wheat variety with those of standard commercial wheat in patients with known NCGS. We performed a double-blind randomized cross-over trial of 42 patients (mean age 45 years, 8 men) with NCGS diagnosed according to the Salerno criteria and adherence to GFD for at least 12 weeks from screening. Enrolled subjects were randomly assigned to one the following groups of treatment: (A) a two-week diet with Senatore Cappelli wheat variety pasta; (B) a two-week diet with standard commercial pasta. Then, after a two-week washout period on gluten-free diet, each patient crossed over to the other treatment group. Symptoms were assessed through a modified version of the Gastrointestinal Symptom Rating Scale (GSRS), tailored on NCGS. Between April 2018 and July 2018, 42 patients with NCGS were enrolled in the study (70.6% females), and 34 patients completed the study. Patients reported lower overall symptoms scores after eating Senatore Cappelli pasta than standard pasta ( p = 0.03) and also significantly lower scores in several specific gastrointestinal and extra-intestinal symptoms after eating Senatore Cappelli pasta than standard pasta, specifically, bloating ( p = 0.04), abdominal distention ( p = 0.004), eructation ( p = 0.01), flatus ( p = 0.02), feeling of incomplete evacuation ( p = 0.001), dermatitis ( p = 0.01), and limb numbness ( p = 0.03). In our study, patients with NCGS experienced lower gastrointestinal and extra-intestinal symptom scores after eating the Senatore Cappelli wheat variety than a standard commercial wheat. Should our preliminary results be confirmed by further studies, new dietary alternatives may be available to patients with NCGS, with consequent health, economic, and social benefits.

The separation of enantiomers has been started in the past and continues to be a topic of great interest in various fields of research, mainly because these compounds could be involved in biological processes such as, for example, those related to human health. Great attention has been devoted to studies for the analysis of enantiomers present in food products in order to assess authenticity and safety. The separation of these compounds can be carried out utilizing analytical techniques such as gas chromatography, high-performance liquid chromatography, supercritical fluid chromatography, and other methods. The separation is performed mainly employing chromatographic columns containing particles modified with chiral selectors (CS). Among the CS used, modified polysaccharides, glycopeptide antibiotics, and cyclodextrins are currently applied.

Analysis of carotenoids is very complex and demanding in terms of both separation and detection. In this article, an analytical strategy relying on high-performance liquid chromatography-photodiode array detection-tandem mass spectrometry (HPLC-PAD-MS/MS) is presented for the large-scale screening of these phytochemicals. Separation was realized by means of Non-Aqueous Reversed Phase (NARP) chromatography on a triacontyl stationary phase kept at subambient temperature, using a mobile phase compatible with atmospheric pressure chemical ionization (APCI). The standards of 14 analytes were used to optimize the method and to predict the chromatographic behaviour of untargeted carotenoids. MS and MS/MS data, obtained during Information Dependent Acquisition (IDA) experiments, were utilized to set up a sensitive HPLC- selected reaction monitoring (SRM) method. Relative abundance between SRM ion currents (ion ratio) allowed the MS distinction of structural isomers (for example, bicyclic, monocyclic and acyclic isomers), while the identification of geometrical isomers was based on Q ratio and fine structure, as calculated from UV-vis spectra. The comparison of LC-PAD- SRM chromatograms, acquired after applying two different extraction procedures (matrix solid-phase dispersion and overnight cold saponification), allowed verifying that sweet peppers are a good source of xanthophylls, prevalently occurring as esterified forms. The overall strategy could identify more than 40 carotenoids in some sweet pepper varieties (Jolly, horn and sweet chili pepper) available on the Italian and European food market., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The separation of chiral compounds is an interesting and important topic of research because these compounds are involved in some biological processes, fundamentally in human health. Among the various application fields where enantiomers are remarkable, drug analysis has to be considered. Most of the drugs contain enantiomers and very often one of the two isomers could be pharmacologically more active or even dangerous. Therefore, the separation of these compounds is very important. Among the different analytical techniques usually employed, capillary electrochromatography has demonstrated great capability in enantiomers resolution. The great potential of this electromigration technique stands mainly in its high efficiency due to the use of an electrosmotic flow (flat flow profile) and on the high selectivity because of the use of a stationary phase. Chiral separation can be obtained utilizing several chiral stationary phases including a polysaccharide derivative. The aim of this review paper is to summarize the main features of capillary electrochromatography and polysaccharide derivatives of chiral stationary phase. It also report examples of practical applications utilizing this approach., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

1.8 μm Silica hydride particles have been derivatized with vancomycin and applied to the enantioseparation of some racemic herbicides and nonsteroidal anti-inflammatory drugs (NSAIDs) by nano-liquid chromatography. The chiral stationary phase (CSP) was packed for only 11 cm and the enantiomers were separated utilizing a laboratory-assembled instrumentation. The new CSP was very effective for the separation of the above mentioned acidic compounds, while poor resolutions were obtained for basic compounds. Mixtures of acetate buffer with methanol or acetonitrile allowed the chiral resolution of all compounds. Fast chiral separation of a NSAIDs-related compound can be achieved in less than 60 s.

The analysis of phenolic compounds in extra virgin olive oils was carried out by high-performance liquid chromatography utilizing photodiode array and mass spectrometry detectors. The chromatographic profile of thirty samples from four Italian Regions highlighted the presence of secoiridoids, phenolic alcohols, flavonoids, and phenolic acid classes. A similar qualitative profile was observed with some differences in peak area and fifteen compounds were tentatively identified. Quantitative analysis was performed by UV detection considering eight standard phenolic compounds. The chromatographic method, after optimization, was validated studying some parameters, e.g., intra-day and inter-day retention time precision, limit of detection, limit of quantification, and linearity. Recovery of the method was performed achieving good results (10 and 50 g·g -1 with recovery of 72.9⁻92.1% ( w / w ) and 79.1⁻102.8% ( w / w ), respectively). In all samples secoiridoids were the main compounds ranging from 85 to more than 99% ( w / w ) of the total concentration of detected phenolic compounds while phenolic acids accounted for the lowest percentage (0.1⁻0.6%, w / w ). Finally, total concentration of phenolic compounds and antioxidant activity were determined with different chemical assays. A good and significant correlation among total phenolic compound concentration and antioxidant activity was observed. A significant different phenolic compound concentration and antioxidant activity was determined between samples from Puglia and Sicily. This was studied performing statistical analysis by one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test.

In this study our preliminary attempt for obtaining fast and highly efficient separations of enantiomers in high-performance liquid chromatography with slightly modified state-of-the-art commercial instrumentation is described. In order to reach this goal after careful selection of chiral analytes, the preparation of chiral stationary phase (CSP), mobile phase composition and column dimensions were optimized. The concept of segmented chiral-achiral column was introduced. As the result of these optimizations baseline separation of enantiomers was achieved with the analysis time between 1-2 s., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hazelnut kernel phenolic compounds were recovered applying two different extraction approaches, namely ultrasound-assisted solid/liquid extraction (UA-SLE) and solid-phase extraction (SPE). Different solvents were tested evaluating total phenolic compounds and total flavonoids contents together to antioxidant activity. The optimum extraction conditions, in terms of the highest value of total phenolic compounds extracted together to other parameters like simplicity and cost were selected for method validation and individual phenolic compounds analysis. The UA-SLE protocol performed using 0.1 g of defatted sample and 15 mL of extraction solvent (1 mL methanol/1 mL water/8 mL methanol 0.1% formic acid/5 mL acetonitrile) was selected. The analysis of hazelnut kernel individual phenolic compounds was obtained by HPLC coupled with DAD and MS detections. Quantitative analysis was performed using a mixture of six phenolic compounds belonging to phenolic classes' representative of hazelnut. Then, the method was fully validated and the resulting RSD% values for retention time repeatability were below 1%. A good linearity was obtained giving R 2 no lower than 0.997.The accuracy of the extraction method was also assessed. Finally, the method was applied to the analysis of phenolic compounds in three different hazelnut kernel varieties observing a similar qualitative profile with differences in the quantity of detected compounds., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pressurized liquid extraction (PLE) has been used for the first time in this work to extract phenolic compounds from Goji berries according to a multilevel factorial design using response surface methodology. The global yield (% w/dw, weight/dry-weight), total phenolic content (TPC), total flavonoid (TF) and antioxidant activity (determined via ABTS assay, expressed as TEAC value) were used as response variables to study the effects of temperature (50-180°C) and green solvent composition (mixtures of ethanol/water). Phenolic compounds characterization was performed by high performance liquid chromatography-diode array detector-tandem mass spectrometry (HPLC-DAD-MS/MS). The optimum PLE conditions predicted by the model were as follows: 180°C and 86% ethanol in water with a good desirability value of 0.815. The predicted conditions were confirmed experimentally and once the experimental design was validated for commercial fruit samples, the PLE extraction of phenolic compounds from three different varieties of fruit samples (Selvatico mongolo, Bigol, and Polonia) was performed. Nine phenolic compounds were tentatively identified in these extracts, including phenolic acids and their derivatives, and flavonols. The optimized PLE conditions were compared to a conventional solid-liquid extraction, demonstrating that PLE is a useful alternative to extract phenolic compounds from Goji berry., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The analysis of pomegranate phenolic compounds belonging to different classes in different fruit parts was performed by high-performance liquid chromatography coupled with photodiode array and mass spectrometry detection. Two different separation methods were optimized for the analysis of anthocyanins and hydrolyzable tannins along with phenolic acids and flavonoids. Two C 18 columns, core-shell and fully porous particle stationary phases, were used. The parameters for separation of phenolic compounds were optimized considering chromatographic resolution and analysis time. Thirty-five phenolic compounds were found, and 28 of them were tentatively identified as belonging to four different phenolic compound classes; namely, anthocyanins, phenolic acids, hydrolyzable tannins, and flavonoids. Quantitative analysis was performed with a mixture of nine phenolic compounds belonging to phenolic compound classes representative of pomegranate. The method was then fully validated in terms of retention time precision, expressed as the relative standard deviation, limit of detection, limit of quantification, and linearity range. Phenolic compounds were analyzed directly in pomegranate juice, and after solvent extraction with a mixture of water and methanol with a small percentage of acid in peel and pulp samples. The accuracy of the extraction method was also assessed, and satisfactory values were obtained. Finally, the method was used to study identified analytes in pomegranate juice, peel, and pulp of six different Italian varieties and one international variety. Differences in phenolic compound profiles among the different pomegranate parts were observed. Pomegranate peel samples showed a high concentration of phenolic compounds, ellagitannins being the most abundant ones, with respect to pulp and juice samples for each variety. With the same samples, total phenols and antioxidant activity were evaluated through colorimetric assays, and the results were correlated among them.

The present study reports successful separations of enantiomers of selected chiral sulfoxides with very high separation factor in high-performance liquid chromatography by using chiral columns prepared with the chiral selector cellulose tris(4-chloro-3-methylphenylcarbamate). High separation factors were observed in polar organic, as well as in hydrocarbon-alcohol-type mobile phases. The key structural components of the solute for obtaining high chiral recognition are discussed as well as thermodynamic quantities of analyte adsorption on the chiral stationary phase were determined. Experiment aimed at the enantioselective extraction of racemates from solution are also described., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This review gives an overview of the phenolic compounds composition of cocoa beans and their modification during manufacturing processes to the final products. Recently published papers dealing with the qualitative and quantitative analysis of the different classes of cocoa phenolic compounds will be discussed. Modifications of the qualitative profile and amount of phenolic compounds in cocoa after the main processes of production chain, fermentation, drying, roasting, and alkalization, will be described. The second part will focus on some of the biological effects described for cocoa phenolic compounds in vitro and in vivo. In particular, the effects of cocoa flavanols on cardiovascular health and endothelial function have been extensively investigated over the last decades, with interesting results from nutritional intervention trials and molecular studies. A few recent updates on the role of cocoa and chocolate consumption on sport performances will be reported., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

A new experimental chiral stationary phase containing amylose 3,5-dimethylphenylcarbamate immobilized onto silica gel (i-ADMPC) was packed in 100μm I.D. fused silica capillary and used for the chiral separation of eight selected flavanone derivatives in polar organic mobile phase by using different miniaturized techniques, such as nano-liquid chromatography (nano-LC), capillary electrochromatography (CEC) and pressure-assisted CEC (pCEC). A comparative study of different elution modes in terms of chromatographic efficiency, analysis time and enantiomeric resolution was carried out. In pCEC mode, the highest chromatography performance was obtained applying +2.5kV voltage and inlet pressure 10bar. Under these conditions, the analysis times were shorter than 20min, and chromatographic efficiencies were in the range 33,000-49,000 plates/m (first eluted peak). The solvent versatility of packed i- ADMPC capillary column was also investigated. In nano-LC, the CSP was stressed under the use of strong organic solvents such as ethyl acetate, acetone and methyl t-butyl ether (MTBE). After at least 40 working hours and over 50 sample injections, the CSP resulted to be very stable allowing to achieve good repeatability employing again the mobile phase containing polar organic solvent. Enantioresolutions and chromatographic efficiencies decreased by 1.60 and 10%, respectively as the result of using above mentioned strong solvents., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Exosomes are involved in intercellular communication. We previously reported that sodium butyrate-induced differentiation of HT29 colon cancer cells is associated with a reduced CD133 expression. Herein, we analyzed the role of exosomes in the differentiation of HT29 cells. Exosomes were prepared using ultracentrifugation. Gene expression levels were evaluated by real-time PCR. The cell proliferation rate was assessed by MTT assay and with the electric cell-substrate impedance sensing system, whereas cell motility was assessed using the scratch test and confocal microscopy. Sodium butyrate-induced differentiation of HT29 and Caco-2 cells increased the levels of released exosomes and their expression of CD133. Cell differentiation and the decrease of cellular CD133 expression levels were prevented by blocking multivesicular body maturation. Exosomes released by HT29 differentiating cells carried increased levels of miRNAs, induced an increased proliferation and motility of both colon cancer cells and normal fibroblasts, increased the colony-forming efficiency of cancer cells, and reduced the sodium butyrate-induced differentiation of HT29 cells. Such effects were associated with an increased phosphorylation level of both Src and extracellular signal regulated kinase proteins and with an increased expression of epithelial-to-mesenchymal transition-related genes. Release of exosomes is affected by differentiation of colon cancer cells; exosomes might be used by differentiating cells to get rid of components that are no longer necessary but might continue to exert their effects on recipient cells., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Imbalanced dietary n-3 and n-6 PUFA content has been associated with a number of neurological conditions. Endocannabinoids are n-6 PUFA derivatives, whose brain concentrations are sensitive to modifications of fatty acid composition of the diet and play a central role in the regulation of mood and cognition. As such, the endocannabinoid system appears to be an ideal candidate for mediating the effects of dietary fatty acids on mood and cognition. Lifelong administration of isocaloric α-linolenic acid (ALA)-deficient and -enriched diets induced short-term memory deficits, whereas only dietary ALA enrichment altered emotional reactivity in adult male rats compared with animals fed a standard diet that was balanced in ALA/linoleic acid (LA) ratio. In the prefrontal cortex, both diets reduced 2-AG levels and increased MAG lipase expression, whereas only the enriched diet reduced AEA levels, simultaneously increasing FAAH expression. In the hippocampus, an ALA-enriched diet decreased AEA content and NAPE-PLD expression, and reduced 2-AG content while increasing MAG lipase expression. These findings highlight the importance of a diet balanced in fatty acid content for normal brain functions and to support a link between dietary ALA, the brain endocannabinoid system, and behavior, which indicates that dietary ALA intake is a sufficient condition for altering the endocannabinoid system in brain regions modulating mood and cognition., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Polyphenols-rich cocoa has many beneficial effects on human health, such as anti-inflammatory effects. Macrophages function as control switches of the immune system, maintaining the balance between pro- and anti-inflammatory activities. We investigated the hypothesis that cocoa polyphenol extract may affect macrophage proinflammatory phenotype M1 by favoring an alternative M2 anti-inflammatory state on macrophages deriving from THP-1 cells. Chemical composition, total phenolic content, and antioxidant capacity of cocoa polyphenols extracted from roasted cocoa beans were determined. THP-1 cells were activated with both lipopolysaccharides and interferon- γ for M1 or with IL-4 for M2 switch, and specific cytokines were quantified. Cellular metabolism, through mitochondrial oxygen consumption, and ATP levels were evaluated. Here, we will show that cocoa polyphenolic extract attenuated in vitro inflammation decreasing M1 macrophage response as demonstrated by a significantly lowered secretion of proinflammatory cytokines. Moreover, treatment of M1 macrophages with cocoa polyphenols influences macrophage metabolism by promoting oxidative pathways, thus leading to a significant increase in O 2 consumption by mitochondrial complexes as well as a higher production of ATP through oxidative phosphorylation. In conclusion, cocoa polyphenolic extract suppresses inflammation mediated by M1 phenotype and influences macrophage metabolism by promoting oxidative pathways and M2 polarization of active macrophages.

Objective: This retrospective study aimed to evaluate the role of NFKB1-94 insertion/deletion ATTG (rs28362491) and NFkBIA 2758 A>G (rs696) polymorphisms and HLA-Cw6 allele in predicting the response to etanercept, a TNF-α blocker, in a population of psoriatic patients naive to biologics., Methods: Genomic DNA was extracted from whole blood in a series of 96 psoriatic patients who received etanercept for at least 3 months. Patients were classified as responders if they achieved a Psoriasis Area and Severity Index improvement of at least 75% after 12 weeks of etanercept treatment and as nonresponders if Psoriasis Area and Severity Index improvement was less than 75%. Genotyping was performed using the PCR-restriction fragment length polymorphism (PCR-RFLP) method., Results: We did not find any significant role of NFKB1-94 insertion/deletion ATTG (rs28362491) and NFkBIA 2758 A>G (rs696) polymorphisms and the HLA-Cw6 allele in predicting the response to etanercept., Conclusion: Our findings suggest that NFKB1 and NFkBIA polymorphisms are not related to the response to etanercept. They also indicate that the therapeutic response to etanercept is not influenced by the presence of the HLA-Cw6 allele, in contrast with previous evidence on ustekinumab, suggesting that such an association is related more to drug than to disease characteristics.

Chemical composition of pomegranate juice can vary due to cultivar, area of cultivation, ripening, climate, and other variables. This study investigates the polyphenolic composition and antioxidant activity of juices obtained from six old Italian pomegranate cultivars. Fruit accessions physicochemical characteristics were determined. Total polyphenols content (TPC), anthocyanin content (TAC) and proanthocyanidin content (TPAC) were measured in the juice samples. Phenolic bioactive molecules were analyzed by HPLC-photodiode array (PDA)/ESI-MS in all the pomegranate juices. In total, seven nonanthocyanidinic and six anthocyanidinic compounds were identified. The six anthocyanins were found in all juices although at different amounts. These results were correlated with antioxidant activity measured by three different chemical assays: 2,2 diphenyl-1-picrylhydrazyl (DPPH(•) ) scavenging activity assay, Trolox equivalent antioxidant capacity (TEAC) method and ferric reducing-antioxidant power (FRAP) assay. Pomegranate juices obtained by six different varieties show variable polyphenolic content and antioxidant activity. The antioxidant capacity methods used have shown variable sensitivity, supporting the hypothesis that different methods for the assessment of antioxidant capacity of food compounds are indeed necessary, due to complexity of sample composition and assay chemical mechanism and sensitivity. Juices from Italian pomegranate show good levels of polyphenols content and antioxidant activity making them potential candidates for employment in the food industry., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A novel vancomycin silica hydride stationary phase was synthesized and the particles of 1.8 µm were packed into fused silica capillaries of 75 µm internal diameter (I.D.). The chiral stationary phase (CSP) was tested for the separation of some derivatized amino acid enantiomers by using nano-liquid chromatography (nano-LC). Some experimental parameters such as the type and the content of organic modifier, the pH, and the concentration of the buffer added to the mobile phase were modified and the effect on enantioselectivity, retention time, and enantioresolution factor was studied. The separation of selected dansyl amino acids (Dns-AAs), e.g., Asp, Glu, Leu, and Phe in their enantiomers was initially achieved utilizing a mobile phase containing 85% (v/v) methanol (MeOH) and formate buffer measuring the enantioresolution factor and enantioselectivity in the range 1.74-4.17 and 1.39-1.59, respectively. Better results were obtained employing a more polar organic solvent as acetonitrile (ACN) in the mobile phase. Optimum results (Rs 1.41-6.09 and α 1.28-2.36) were obtained using a mobile phase containing formate buffer pH 2.5/water/MeOH/ACN 6:19:12.5:62.5 (v/v/v/v) in isocratic elution mode at flow rate of 130 nL/min., (© 2015 Wiley Periodicals, Inc.)

A chemical characterization of major lipid components, namely, triacylglycerols, fatty acids and the unsaponifiable fraction, in a Quinoa seed lipids sample is reported. To tackle such a task, non-aqueous reversed-phase high-performance liquid chromatography with mass spectrometry detection was employed. The latter was interfaced with atmospheric pressure chemical ionization for the analysis of triacylglycerols. The main triacylglycerols (>10%) were represented by OLP, OOL and OLL (P = palmitoyl, O = oleoyl, L = linoleoyl); the latter was present in the oil sample at the highest percentage (18.1%). Furthermore, fatty acid methyl esters were evaluated by gas chromatography with flame ionization detection. 89% of the total fatty acids was represented by unsaturated fatty acid methyl esters with the greatest percentage represented by linoleic and oleic acids accounting for approximately 48 and 28%, respectively. An extensive characterization of the unsaponifiable fraction of Quinoa seed lipids was performed for the first time, by using comprehensive two-dimensional gas chromatography with dual mass spectrometry/flame ionization detection. Overall, 66 compounds of the unsaponifiable fraction were tentatively identified, many constituents of which (particularly sterols) were confirmed by using gas chromatography with high-resolution time-of-flight mass spectrometry., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A method for the analysis of flavonoids (myricetin, quercetin, naringenin, hesperitin, and kaempferol), with interesting bioactivity, has been developed and validated utilizing nano-LC technique. In order to find optimal conditions, capillary columns (75 μm id × 10 cm) packed with different types of stationary phases, Kinetex® C18 core-shell (2.6 μm particle size), Hydride-based RP-C18 (sub-2 μm particle size), and LiChrospher® 100 RP-18 endcapped (5 μm particle size) were evaluated. The method was validated using Hydride-based RP-C18 stationary phase, with sub-2 μm particle size. A good chromatographic performance, expressed in terms of repeatability (RSD, in the range 1.63-4.68% for peak area), column-to-column reproducibility (RSD not higher than 8.01% for peak area), good linearity and sensitivity was obtained. In particular limit of detection values between 0.07 and 0.31 μg/mL were achieved with on column focusing technique. The method was applied to the determination of studied flavonoids in dietary supplements as well as in food matrices. The amount of quercetin found in the first analyzed dietary supplement, was in agreement to the labeled content. In the other samples, where the content of flavonoids was not labeled, most of the studied flavonoids were determined in amounts somewhere comparable to those reported in literature., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

It has been hypothesized that inflammatory response triggered by surgery might induce the release of molecules that could promote proliferation, invasion and metastasis of surviving cancer cells. To test this hypothesis, the levels of multiple inflammation-related circulating factors were analyzed in patients undergoing surgery for colorectal cancer. A Luminex xMAP system was used to simultaneously assess levels of IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, FGF, eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α and VEGF in 20 colorectal cancer patients and 10 age-matched non-neoplastic patients. In cancer patients analyses were performed at baseline (before surgery) and at different time points (up to 30 days) following laparoscopic surgery. Significantly higher levels of IL-1β, IL-7, IL-8, G-CSF, IFN-γ and TNF-α were detected in colorectal cancer patients compared to controls at baseline. In colorectal cancer patients, circulating levels decreased progressively following surgery and after day 30 post-surgery were no longer different from controls. These findings suggest that expression levels of several cytokines are higher in colorectal cancer patients compared to control subjects and no significant increase in several inflammation-related circulating factors is observed following laparoscopic surgery for cancer. Confirmation and validation in a different and larger cohort of patients are warranted.

In this study, a simple and rapid methodology to analyze and quantify principal flavanones in citrus fruit juices through the use of a nano-LC/UV-Vis apparatus, employing a 75 μm id capillary column packed with sub-2 μm particles C18 stationary phase for 10 cm, was developed. All compounds were baseline resolved working with a step gradient elution mode in 10 min. The developed analytical method was validated and the resulting RSD% for intra- and interday repeatability, related to retention time and peak area, were <4.7 and 5.5%, respectively. LOD and LOQ values corresponded to 0.40 and 1.56 μg/mL for didymin, hesperitin, and narinegenin, while for the other flavanones were 0.78 and 3 μg/mL, respectively. Good linearity with acceptable determination coefficients R(2) was obtained in the range between LOQ concentration and 200 μg/mL (500 μg/mL naringin and hesperidin). Good recovery values were also obtained. Then, the method was applied to the analysis of selected hand-squeezed and commercial citrus juices. Further, the nano-LC system was coupled to a mass spectrometer to confirm analyte identification. Antioxidant capacity of selected samples was also evaluated measured by Folin-Ciocalteu assay and Trolox equivalent antioxidant capacity assay. Results were compared to determine total flavanones concentrations., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Colorectal cancer remains one of the most common and lethal malignancies worldwide despite the use of various therapeutic strategies. A better understanding of the mechanisms responsible for tumor initiation and progression is essential for the development of novel, more powerful therapies. The traditional, so-called "stochastic model" of tumor development, which assumes that each cancer cell is tumorigenic, has been deeply challenged during the past decade by the identification of cancer stem cells (CSCs), a biologically distinct subset of cells within the bulk of tumor mass. This discovery led to the development of the hierarchical model of tumorigenesis which assumes that only CSCs have the ability to initiate tumor growth, both at primary and metastatic sites. This model implies that the elimination of all CSCs is fundamental to eradicate tumors and that failure to do so might be responsible for the occurrence of relapses and/or metastases frequently observed in the clinical management of colorectal cancer patients. Identification and isolation of CSCs is essential for a better understanding of their role in the tumorigenetic process and for the development of CSC-specific therapies. Several methods have been used for this purpose and many efforts have been focused on the identification of specific CSC-surface markers. This review provides an overview of the proposed roles of CSC in human colorectal tumorigenesis focusing on the most important molecules identified as CSC-specific markers in colorectal cancer and on the potential strategies for the development of CSC-targeted therapy.

In this study, a nano-liquid chromatography based method for the simultaneous separation of 16 polyphenols employing UV-vis detection has been developed. A 100 μm I.D. capillary column packed with C18 core-shell particles (2.6 μm particle size, 100 Å) for 10 cm was employed. The separation of analytes was performed with a step gradient in less than 20 min, using 0.5% formic acid aqueous solution and acetonitrile as eluents. The optimized analytical method was validated and the resulting RSD% for intra-day and inter-day repeatability, related to retention time, retention factor and peak area, were below 4.68 and 5.57%, respectively. LOD and LOQ values were as low as 0.78 and 3.12 μg/mL, while linearity, assessed in the concentration range of interest for all analytes, gave R(2)≥0.990. The method was finally applied to the analysis of polyphenols extracted from a collected bee pollen. Nine polyphenols, namely o-, p-coumaric acid, ferulic acid, myricetin, cinnamic acid, quercetin, naringenin, hesperitin and kaempferol, were identified. All analytes, with the exception of p-coumaric acid and myricetin, which partially co-eluted with other pollen components, were also quantified in the sample., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Candida mannan (Mn) detection in bronchoalveolar lavage fluid (BALF) was shown to be useful for earlier identification and preemptive therapy targeting in preterm infants at high risk of invasive Candida infection. We investigated whether early detection of Candida Mn in BALF is associated with the presence of some neutrophilic products, as markers of prenatal infection/inflammation., Methods: BALF specimens were collected during the first 48 h of life from mechanically ventilated preterm newborns. Samples were analyzed by high-performance liquid chromatography-electrospray ionization-mass spectrometry. The relative amounts of α-defensins 1-4 and S100A proteins were measured by extracted ion current peak area. Absolute and differential white cell counts in BALF were obtained. Mn antigen concentrations were determined by the Platelia Candida antigen kit., Results: Twenty-five studied neonates were divided into two groups: Mn-positive group and Mn-negative group. Levels of α-defensins 1-4 and S100A12 were significantly higher in the Mn-positive group than in the Mn-negative group. Moreover, positive significant correlations between the absolute number of neutrophils and the levels of α-defensins 1-4 and S100A8 were observed., Conclusion: The detection of Mn antigen in BALF of preterm infants is consistent with evidence of an innate immune response in their lungs as demonstrated by higher levels of α-defensins and S100A proteins.

Graft-versus-host disease (GVHD) is the major life-threatening complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), developing in 35%-70% of all allo-HSCT recipients despite immunosuppressive prophylaxis. The recent application of proteomic tools that allow screening for differentially expressed or excreted proteins in body fluids could possibly identify specific biomarkers for GVHD. Whole saliva is highly attractive for noninvasive specimen collection. In the present study, we collected saliva specimens from 40 consecutives patients who underwent allo-HSCT between December 2008 and March 2011 at our institution. The specimens were analyzed by HPLC coupled to electrospray-ionization mass spectrometry. Variable expression of S100 protein family members (S100A8, S100A9, and S100A7) was detected. Fourteen of 23 patients with GVHD demonstrated the presence of S100A8, compared with only 2 patients without GVHD and 0 patients in the control group (P = .001). S100A7 was detectable in 11 of the 23 patients with GVHD but was absent in the other 2 groups (P = .0001). S100A9-short was detected in 20 patients with GVHD, in 9 patients without GVHD, and in 8 healthy volunteers (P = .01) Further studies are needed to clarify the role of these proteins as a marker of GVHD or as an index of mucosal inflammation., (Copyright © 2013 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

High-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) mass -spectrometry (MS) is a relevant technique for the detection and relative quantitation of naturally occurring peptides and proteins. The peptide/protein mass is determined by deconvolution of the ESI-MS spectrum, and the resolution can be better than 1:10,000 with the instruments currently available. Accurate mass measurement, coupled with sufficient resolution, makes it possible to greatly restrict the enormous number of possible molecular formulas that might be represented by a specific molecular mass. As soon as the protein mass has been unequivocally attributed to a specific structure by means of different enzymatic and chemical treatments, the m/z values detected in the ESI spectrum can be utilized to reveal the protein and to perform its relative quantitation, by the extracted ion current (XIC) procedure, in an unlimited number of samples. This chapter describes the HPLC-ESI-MS experimental conditions which allow detecting and quantifying-in human saliva-different S100 proteins and their isoforms.

Nano-LC and CEC were studied for the separation of cytochrome c tryptic digest. The peptides mixture was analyzed using either a nano-LC commercial or a laboratory assembled instrumentation coupled with an IT-ESI-MS by using a nanospray interface. CEC experiments were carried out with a CE apparatus coupled with the IT-ESI-MS through a liquid junction interface. Analytes were separated utilizing C18 silica based stationary phases, of different properties and origin, silica derivatized with cyano groups and C18 monolithic material. The last column, just because the chemical composition (absence of charged/chargeable groups) was tested only using nano-LC. Best results mainly related to the highest number of peptides separated and column equilibration time were obtained by nano-LC employing the C18 stationary phase (detection of 20 peptides, coverage of 88%). Similar results were achieved using both commercial and laboratory assembled instrumentation. The use of CEC revealed a higher separation efficiency and shorter analysis time. However, the number of separated peptides were lower than those observed in nano-LC. In CEC the use of capillaries packed with cyanosilica particles offered better results; however, less satisfactory than those observed in the miniaturized LC technique. Provided the use of the same stationary phase and taking into account the driving forces, the two techniques can be considered complementary, offering different information related to the retention times of the studied peptides., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, a rapid separation of eleven polyphenols and three methylxanthines was obtained by means of nano-liquid chromatography (nano-LC), employing a 100 μm I.D. capillary column packed with C18 core-shell particles (2.7 μm) for 10 cm. All compounds were baseline resolved with a step gradient elution in less than 15 min. The developed analytical method was validated and the resulting RSD% for intra-day and inter-day repeatability, related to retention time, retention factor and peak area, were below 5.1 and 5.7%. LOD and LOQ values corresponded to 0.300 and 0.625 μg/mL, while linearity range assessed gave R² no lower than 0.990. Then, the method was used to determine studied compounds in tea extracts. Further, the nano-LC system was coupled with a mass spectrometer to confirm the components present in real samples. Finally the results were compared with those obtained using a capillary column (100 μm I.D. × 10 cm) packed with C18 sub-2 μm particles, applying the same nano-LC experimental conditions., (Copyright © 2012 Elsevier B.V. All rights reserved.)

An HPLC-ESI-MS analysis of adult human whole saliva evidenced three protein masses (M average 11,487±2, 11,301±2 and 22,362±3Da) eluting in the 32.5-35.0min range. Treatment in reducing conditions allowed establishing that they are S-derivatives of N-terminal acetylated cystatin B, namely its S-glutathionyl, S-cysteinyl and S-S dimer. The identification was confirmed by high resolution HPLC-ESI-MS-MS experiments on the intact naturally occurring proteins and their tryptic digests. S-unmodified cystatin B is rarely detectable in whole saliva of healthy adults (5 subjects out of 65) and its percentage does not overcome approximately 20% of total cystatin B (11±9%). In the majority of subjects (60 out of 65) the mean percentages of the S-modified derivatives were S-glutathionyl 53±13%, S-cysteinyl 15±5%, S-S 2-mer 32±13%. Variations of the percentages of these S-modified derivatives of cystatin B could be indicative of oral oxidative stress. As we are aware, this is the first time that S-glutathionylation and S-cysteinylation were described as extensive PTM of a salivary protein and the first time that these PTMs were detected in naturally occurring cystatin B., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The development of new separation techniques and different mass spectrometry instrumental devices, as well as the great availability of specific reactants, offers ample choice to scientists for carrying out high-throughput proteomic studies and being competitive in the field today. However, the different options available often do not provide comparable results, which can be linked to factors such as the strategy adopted, the nature of the sample and the instrumental availability. In this critical review, the results obtained so far in the study of human saliva by different proteomic approaches will be compared and discussed., (© 2012 Expert Reviews Ltd)

A chemical characterization of the major components, namely, triacylglycerols (TAGs), polyphenols, and tocopherols in a Sacha inchi oil derived from cold pressing of the seed, is hereby reported. To tackle such a task, high-performance liquid chromatography in combination with photodiode array (PDA), fluorescence (RF), and mass spectrometry (MS) detection was employed. The latter was interfaced with atmospheric pressure chemical ionization and with electrospray ionization for the analysis of TAGs and polyphenols, respectively, whereas RF detection was tested for the determination of tocopherol content. Furthermore, fatty acid methyl esters (FAMEs) were evaluated by gas chromatography-flame ionization detector. A 93% amount of total fatty acids was represented by unsaturated FAMEs with the greatest percentage represented by linoleic (L) and linolenic (Ln) accounting for approximately 50 and 36%, respectively. The main TAGs (>10%) were represented by LLnL, LnLnLn, and LnLLn; the latter was present in the oil sample at the highest percentage (22.2%). Among tocopherols, γ-tocopherol was detected to be the most abundant component (over 50%). The polyphenolic composition was also investigated, and a total of 15 compounds were positively identified, through the complementary analytical information coming from PDA and MS data. To the best of our knowledge, this is the first report providing a thorough chemical characterization of a Plukenetia volubilis L. oil.

The use of human saliva as a diagnostic and prognostic fluid has until recently been somewhat disregarded. Although sample collection is non-invasive, physiological and genetic variations were largely responsible for its infrequent application in the past. Recently, several proteomic studies contributed to partial elucidation of the salivary proteome (more than 2400 protein components have been characterized), both in terms of composition, contributions to whole saliva and genetic/physiological variability. On this basis, is not too optimistic to believe that in the near future human saliva could become a relevant diagnostic fluid. In this review, the characterization by proteomic approaches of new salivary markers in oncology, head and neck carcinoma (oral cavity, oropharynx, larynx, and salivary glands), breast and gastric cancers, salivary gland function and disease, Sjögren syndrome, systemic sclerosis, dental and gingival pathology, systemic, psychiatric and neurological diseases, is described.

Background: Urinary lithiasis is one of the most common benign urological diseases. There is growing evidence that a delicate equilibrium regulated by the function of proteins, soluble peptides, membrane proteins and intracellular mechanisms actually exists. We have studied the urinary protein composition of patients affected by calcium oxalate nephrolithiasis in order to discover a biomarker or any predisposing factors., Methods: The urinary protein composition of 17 patients (11 males, 6 females; mean age 45yrs ± 14SD), affected by calcium oxalate nephrolithiasis, was assessed in comparison with 17 healthy subjects. A qualitative assay was performed using MALDI-TOF mass spectrometry in a spectrum between 1 and 5kDa (medium size peptides), and a numerical (quantitative) assay using specific filters and MicroBCA Protein Assay., Results: No differences were detected in the mass spectrums between patients and control subjects: all peaks overlapped perfectly. The results of the numerical assay suggest that concentrations of protein species <5kDa in control samples were actually higher than those which were found in patients. The differences are statistically significant., Conclusions: The study detected neither a biomarker nor any predisposing factors in "stone former" patients. The assessment of the results obtained, in terms of quantitative differences, indicate the need for further research.

LC-MS-based shotgun proteomics relies both on the power of the separation techniques and the sensitivity of detection methods. As a viable alternative to classical approaches in this field, we developed a fully automated, comprehensive 2D LC system, in which RPLC × RPLC was coupled to MS detection, for the first time, and applied for the analysis of tryptic digests obtained from α-casein and dephosphorylated α-casein. The use of a significantly different pH in the two dimensions allowed us to attain high peak capacity, despite the employment of novel identical stationary phases. Furthermore, such a combination addresses compatibility issues, thus allowing straightforward interfacing in online 2D LC configuration, as well as direct linkage to a mass spectrometer. A theoretical peak capacity of ca. 8500 was calculated for the setup, employing four serially coupled C18 columns in the first dimension (600 × 2.1 mm, 2.7 μm d.p.), operated under basic conditions, and 3 cm length of the same stationary phase (30 × 4.6 mm, 2.7 μm d.p. column), under acidic conditions, for fast second dimension analysis.

Background: β-thymosins play roles in cytoskeleton rearrangement, angiogenesis, fibrosis and reparative process, thus suggesting a possible involvement in the pathogenesis of systemic sclerosis. The aim of the study was to investigate the presence of thymosins β4, β4 sulfoxide, and β10 in bronchoalveolar lavage fluid of scleroderma patients with interstitial lung disease and the relation of these factors with pulmonary functional and radiological parameters., Methods: β-thymosins concentrations were determined by reverse phase-high performance liquid chromatography-electrospray-mass spectrometry in the bronchoalveolar lavage fluid of 46 scleroderma patients with lung involvement and of 15 controls., Results: Thymosin β4, β4 sulfoxide, and β10 were detectable in bronchoalveolar lavage fluid of patients and controls. Thymosin β4 levels were significantly higher in scleroderma patients than in controls. In addition, analyzing the progression of scleroderma lung disease at one-year follow-up, we have found that higher thymosin β4 levels seem to have a protective role against lung tissue damage. Thymosin β4 sulfoxide levels were higher in the smokers and in the scleroderma patients with alveolitis., Conclusions: We describe for the first time β-thymosins in bronchoalveolar lavage fluid and their possible involvement in the pathogenesis of scleroderma lung disease. Thymosin β4 seems to have a protective role against lung tissue damage, while its oxidation product mirrors an alveolar inflammatory status.

Nano-LC and conventional HPLC techniques were applied for the analysis of anthocyanins present in commercial fruit juices using a capillary column of 100 μm id and a 2.1 mm id narrow-bore C(18) column. Analytes were detected by UV-Vis at 518 nm and ESI-ion trap MS with HPLC and nano-LC, respectively. Commercial blueberry juice (14 anthocyanins detected) was used to optimize chromatographic separation of analytes and other analysis parameters. Qualitative identification of anthocyanins was performed by comparing the recorded mass spectral data with those of published papers. The use of the same mobile phase composition in both techniques revealed that the miniaturized method exhibited shorter analysis time and higher sensitivity than narrow-bore chromatography. Good intra-day and day-to-day precision of retention time was obtained in both methods with values of RSD less than 3.4 and 0.8% for nano-LC and HPLC, respectively. Quantitative analysis was performed by external standard curve calibration of cyanidin-3-O-glucoside standard. Calibration curves were linear in the concentration ranges studied, 0.1-50 and 6-50 μg/mL for HPLC-UV/Vis and nano-LC-MS, respectively. LOD and LOQ values were good for both methods. In addition to commercial blueberry juice, qualitative and quantitative analysis of other juices (e.g. raspberry, sweet cherry and pomegranate) was performed. The optimized nano-LC-MS method allowed an easy and selective identification and quantification of anthocyanins in commercial fruit juices; it offered good results, shorter analysis time and reduced mobile phase volume with respect to narrow-bore HPLC., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)