Molecular basis of senescence transmitting in the population of human endometrial stromal cells.

Hormone-regulated proliferation and differentiation of endometrial stromal cells (ESCs) determine overall endometrial plasticity and receptivity to embryos. Previously we revealed that ESCs may undergo premature senescence, accompanied by proliferation loss and various intracellular alterations. Here we focused on whether and how senescence may be transmitted within the ESCs population. We revealed that senescent ESCs may induce paracrine senescence in young counterparts via cell contacts, secreted factors and extracellular vesicles. According to secretome-wide profiling we identified plasminogen activator inhibitor -1 (PAI-1) to be the most prominent protein secreted by senescent ESCs (data are available via ProteomeXchange with identifier PXD015742). By applying CRISPR/Cas9 techniques we disclosed that PAI-1 secreted by senescent ESCs may serve as the master-regulator of paracrine senescence progression within the ESCs population. Unraveled molecular basis of senescence transduction in the ESCs population may be further considered in terms of altered endometrial plasticity and sensitivity to invading embryo, thus contributing to the female infertility curing.

Investigating the oxidative refolding mechanism of Cripto-1 CFC domain.

Using a combined approach based on MS, enzyme digestion and advanced MD studies we have determined the sequential order of formation of the three disulfide bridges of the Cripto-1 CFC domain. The domain has a rare pattern of bridges and is involved in the recognition of several receptors. The bridge formation order is C1-C4, C3-C5, C2-C6, however formation of C1-C4 plays no roles for the formation of the others. Folding is driven by formation of the C3-C5 bridge and is supported by residues lying within the segment delimited by these cysteines. We indeed observe that variants CFC-W123A and CFC-ΔC1/C4, where C1 and C4 are replaced by serines, are able to refold in the same time window as the wild type, while CFC-K132A and CFC-W134A are not. A variant where cysteines of the second and third bridge are mutated to serine, convert slowly to the monocyclic molecule. Data altogether support a mechanism whereby the Cripto-1 CFC domain refolds by virtue of long-range intramolecular interactions that involve residues close to cysteines of the second and third bridge. These findings are supported by the in silico study that shows how distant parts of the molecules come into contact on a long time scale.

Nutritional profiling of Eurasian woodcock meat: chemical composition and myoglobin characterization.

BACKGROUND: Meat from birds is a rich source of proteins for the human diet. In this framework, Eurasian woodcock (Scolopax rusticola L.), a medium-small wading bird hunted as game in many Eurasian countries, is considered one of the best meats for culinary purposes. Since the nutritional composition of Eurasian woodcock meat has not yet been reported, we decided to determine the nutritional profile of S. rusticola meat. RESULTS: Macronutrient components (proteins, lipids and fatty acids) were determined, as well as free and total amino acids, and compared with those of the common pheasant. Eurasian woodcock meat contains high levels of proteins and essential amino acids. The levels of unsaturated fatty acids represent a great contribution to the total lipid amount. Among polyunsaturated fatty acids, linoleic acid (C18:2, n-6) is the major essential fatty acid. Finally, we report the characterization of myoglobin (Mb) from Eurasian woodcock. CONCLUSION: The data revealed that meat from this bird could be a good source of quality raw proteins because of its amino acid composition, and it had a low lipid content. On the other hand, Mb characterization might be of benefit to the meat industry, by providing useful information for the determination of species-specific differences in meat from birds. © 2018 Society of Chemical Industry.

Extracellular Vesicles in Bile as Markers of Malignant Biliary Stenoses.

BACKGROUND & AIMS: Algorithms for diagnosis of malignant common bile duct (CBD) stenoses are complex and lack accuracy. Malignant tumors secrete large numbers of extracellular vesicles (EVs) into surrounding fluids; EVs might therefore serve as biomarkers for diagnosis. We investigated whether concentrations of EVs in bile could discriminate malignant from nonmalignant CBD stenoses. METHODS: We collected bile and blood samples from 50 patients undergoing therapeutic endoscopic retrograde cholangiopancreatography at university hospitals in Europe for CBD stenosis of malignant (pancreatic cancer, n = 20 or cholangiocarcinoma, n = 5) or nonmalignant (chronic pancreatitis [CP], n = 15) origin. Ten patients with CBD obstruction due to biliary stones were included as controls. EV concentrations in samples were determined by nanoparticle tracking analyses. The discovery cohort comprised the first 10 patients with a diagnosis of pancreatic cancer, based on tissue analysis, and 10 consecutive controls. Using samples from these subjects, we identified a threshold concentration of bile EVs that could best discriminate between patients with pancreatic cancer from controls. We verified the diagnostic performance of bile EV concentration by analyzing samples from the 30 consecutive patients with a diagnosis of malignant (pancreatic cancer or cholangiocarcinoma, n = 15) or nonmalignant (CP, n = 15) CBD stenosis. Samples were compared using the Mann-Whitney test and nonparametric Spearman correlation analysis. Receiver operating characteristic area under the curve was used to determine diagnostic accuracy. RESULTS: In both cohorts, the median concentration of EVs was significantly higher in bile samples from patients with malignant CBD stenoses than controls or nonmalignant CBD stenoses (2.41 × 1015 vs 1.60 × 1014 nanoparticles/L in the discovery cohort; P < .0001 and 4.00 × 1015 vs 1.26 × 1014 nanoparticles/L in the verification cohort; P < .0001). A threshold of 9.46 × 1014 nanoparticles/L in bile best distinguished patients with malignant CBD from controls in the discovery cohort. In the verification cohort, this threshold discriminated malignant from nonmalignant CBD stenoses with 100% accuracy. Serum concentration of EVs distinguished patients with malignant vs patients with nonmalignant CBD stenoses with 63.3% diagnostic accuracy. CONCLUSIONS: Concentration of EVs in bile samples discriminates between patients with malignant vs nonmalignant CBD stenosis with 100% accuracy. Further studies are needed to confirm these findings. Clinical Trial registration no: ISRCTN66835592.

Trifluoroacetylated tyrosine-rich D-tetrapeptides have potent antioxidant activity.

The term "oxidative stress" indicates a set of chemical reactions unleashed by a disparate number of events inducing DNA damage, lipid peroxidation, protein modification and other effects, which are responsible of altering the physiological status of cells or tissues. Excessive Reactive Oxygen Species (ROS) levels may accelerate ageing of tissues or induce damage of biomolecules thus promoting cell death or proliferation in dependence of cell status and of targeted molecules. In this context, new antioxidants preventing such effects may have a relevant role as modulators of cell homeostasis and as therapeutic agents. Following an approach of peptide libraries synthesis and screening by an ORACFL assay, we have isolated potent anti-oxidant compounds with well-defined structures. Most effective peptides are N-terminally trifluoroacetylated (CF3) and have the sequence tyr-tyr-his-pro or tyr-tyr-pro-his. Slight changes in the sequence or removal of the CF3 group strongly reduced antioxidant ability, suggesting an active role of both the fluorine atoms and of peptide structure. We have determined the NMR solution structures of the active peptides and found a common structural motif that could underpin the radical scavenging activity. The peptides protect keratinocytes from exogenous oxidation, thereby from potential external damaging cues, suggesting their use as skin ageing protectant and as cell surviving agents.

AMP18 interacts with the anion exchanger SLC26A3 and enhances its expression in gastric cancer cells.

AMP18 is a stomach-specific secreted protein expressed in normal gastric mucosa but absent in gastric cancer. AMP18 plays a major role in maintaining gastric mucosa integrity and is characterized by the presence of a BRICHOS domain consisting of about 100 amino acids, present also in several unrelated proteins, and probably endowed with a chaperon-like activity. In this work, we exploited a functional proteomic strategy to identify potential AMP18 interactors with the aim to add knowledge on its functional role within gastric cell lines and tissues. To this purpose, recombinant biotinylated AMP18 was purified and incubated with protein extract from human normal gastric mucosa by applying an affinity chromatography strategy. The interacting proteins were identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. The pool of interacting proteins contained SLC26A3, a protein expressed in the apical membrane of intestinal epithelial cells, supposed to play a critical role in Cl(-) absorption and fluid homeostasis. The interaction was also confirmed by Western blot with anti-SLC26A3 on transfected AGS cell extract following AMP18 pull-down. Furthermore, the interaction between AMP18 and SLC26A3 was also validated by confocal microscopy that showed a co-localization of both proteins at plasma membrane level. More importantly, for the first time, we showed that SLC26A3 is down-regulated in gastric cancer and that the overexpression of AMP18 in AMP-transfected gastric cancer cells up-regulated the expression of SLC26A3 both at transcriptional and translational level, the latter probably through the activation of the MAP kinases pathway. These findings strongly suggest that AMP18 might play an anti-inflammatory role in maintaining mucosal integrity also by regulating SLC26A3 level.

Structural characterization of dioicin 1 from Phytolacca dioica L. gains novel insights into phylogenetic relationships of Phytolaccaceae type 1 RIPs.

Ribosome-inactivating proteins are plant cytotoxic enzymes, also present in fungi, algae and bacteria, mainly known for their ability to inhibit protein synthesis. We previously purified and structurally characterized three type 1 RIPs (PD-S1-3) from Phytolacca dioica seeds and four type 1 RIPs (PD-L1-4) from adult plant leaves. Two additional RIPs, named dioicin 1 and dioicin 2, were isolated from leaves of young plants and developing leaves of adult plants. The evidence that P. dioica synthesizes and accumulates these RIPs isoforms suggests that these proteins have been conserved during evolution. Though several aspects of P. dioica type 1 RIP characterization have been studied, some important questions remain to be answered especially with respect to Phytolaccaceae RIP evolution. One of the major problems encountered in approaching RIPs phylogeny concerns the availability of their sequences. In this study, we report the characterization of biological and structural properties of dioicin 1, including the determination of its primary structure by using a combined approach based on Edman degradation, de novo sequencing by ESI-Q-TOF-MS/MS and peptide mapping by MALDI-TOF MS. Knowledge of dioicin 1 primary structure provide us a mean to deepen Phytolaccaceae's RIPs phylogeny. We speculate that both dioicins 1 and 2 share common ancestors with PAP-II and PAP icos-II and that dioicin 1 is not closely related to other members of this clade, thus shedding lights on evolutionary relationships among type 1 RIPs from Phytolaccaceae.

De-regulated expression of the BRG1 chromatin remodeling factor in bone marrow mesenchymal stromal cells induces senescence associated with the silencing of NANOG and changes in the levels of chromatin proteins.

Stem cells have a peculiar chromatin architecture that contributes to their unique properties, including uncommitted status, multi/pluripotency and self-renewal. We analyzed the effect of the de-regulation of the SWI/SNF chromatin remodeling complex in mesenchymal stromal cells (MSC) through the silencing and up-regulation of BRG1, which is the ATPase subunit of the complex. The altered expression of BRG1 promoted the senescence of MSC with suppression of the NANOG transcription, which is part of the transcriptional circuitry governing stem cell functions. To gain insight on the way NANOG was silenced, we evaluated how the de-regulated BRG1 expression affect the binding of activators and repressors on the NANOG promoter. We found 4 E2F binding motifs on NANOG promoter, which can be occupied by RB1 and RB2/P130. These are members of the retinoblastoma gene family. In MSC with a silenced BRG1, the relative binding of the 2 retinoblastoma proteins increased, and this was associated with the recruitment of DNMT1. This induced the methylation of CpG on the NANOG promoter. Opposingly, when a high level of BRG1 was present, the same E2F binding motifs were docking sites for BRG1, which induced chromatin compaction without CpG methylation but with increased histone deacetylation, associated with the presence of HDAC1 on E2F binding sites. Besides the sharp regulation of the NANOG expression, we evidenced, through proteomic analysis, that the de-regulation of the SWI/SNF function affected the expression of histones and other nuclear proteins involved in "nuclear architecture," suggesting that BRG1 may act as global regulator of gene expression.

A targeted secretome profiling by multiplexed immunoassay revealed that secreted chemokine ligand 2 (MCP-1/CCL2) affects neural differentiation in mesencephalic neural progenitor cells.

Chemokines and cytokines, primarily known for their roles in the immune and inflammatory response, have also been identified as key components of the neurogenic niche where they are involved in the modulation of neural stem cell proliferation and differentiation. However, a complete understanding of the functional role played in neural differentiation and a comprehensive profiling of these secreted molecules are lacking. By exploiting the multiplexing capability of magnetic bead-based immunoassays, we have investigated the changes of the expression levels of a set of chemokines and cytokines released from the pluripotent neural cell line mes-c-myc A1 following its differentiation from a proliferating phenotype (A1P) toward a neural (A1D) phenotype. We found a subset of molecules exclusively released from A1P, whereas others were differentially detected in A1P and A1D conditioned media. Among them, we identified monocyte chemoattractant protein-1/chemokine ligand 2 (MCP-1/CCL2) as a proneurogenic factor able to affect neuronal differentiation of A1 cells as well as of neuroblasts from primary cultures and to induce the elongation and/or formation of neuritic processes. Altogether, data are suggestive of a main role played by the CCL2/CCR2 signaling pathway and in general of the network of secreted cytokines/chemokines in the differentiation of neural progenitor cells toward a neural fate.

An improved UPLC method for the detection of undeclared horse meat addition by using myoglobin as molecular marker.

In 2013, following the scandal of the presence of undeclared horse meat in various processed beef products across the Europe, several researches have been undertaken for the safety of consumer health. In this framework, an improved UPLC separation method has been developed to detect the presence of horse myoglobin in raw meat samples. The separation of both horse and beef myoglobins was achieved in only seven minutes. The methodology was improved by preparing mixtures with different composition percentages of horse and beef meat. By using myoglobin as marker, low amounts (0.50mg/0.50g, w/w; ∼0.1%) of horse meat can be detected and quantified in minced raw meat samples with high reproducibility and sensitivity, thus offering a valid alternative to conventional PCR techniques.

Ultra-high performance liquid chromatography tandem mass spectrometry for the detection of durum wheat contamination or adulteration.

In this work, an ultra-performance liquid chromatography electrospray ionization (UPLC-ESI)-MS/MS methodology based on multiple reaction monitoring (MRM) for the selective and sensitive detection and quantification of durum wheat adulteration has been developed and fully validated. The targeted analysis was performed by monitoring specific transitions at m/z 543.7 > 657.4 and m/z 543.7 > 299.2 of a species-specific marker derived from a tryptic peptide of puroindoline a (Pin-a), a cysteine-rich protein selectively present only in common wheat. In addition, two transitions at m/z 500.4 > 725.4 and m/z 500.4 > 561.9 of a reference peptide belonging to purothionin A-1, present in both species, were also monitored. The calibration curves obtained on binary mixtures with known percentages of common/durum wheat flours showed linearity (coefficient of regression, r ≥ 0.99) over concentrations that ranged between 80 and 1%. The limit of detection (LOD) and limit of quantification (LOQ) for the Pin-a marker in wheat flours were 0.01 and 0.03%, respectively. The identified Pin-a marker was also found to be highly diagnostic for the quantification of common wheat in raw materials (kernels) and processed products (pasta), thus offering new opportunities to assess food authenticity.

Molecular profiling of the Phytophthora plurivora secretome: a step towards understanding the cross-talk between plant pathogenic oomycetes and their hosts.

The understanding of molecular mechanisms underlying host-pathogen interactions in plant diseases is of crucial importance to gain insights on different virulence strategies of pathogens and unravel their role in plant immunity. Among plant pathogens, Phytophthora species are eliciting a growing interest for their considerable economical and environmental impact. Plant infection by Phytophthora phytopathogens is a complex process coordinated by a plethora of extracellular signals secreted by both host plants and pathogens. The characterization of the repertoire of effectors secreted by oomycetes has become an active area of research for deciphering molecular mechanisms responsible for host plants colonization and infection. Putative secreted proteins by Phytophthora species have been catalogued by applying high-throughput genome-based strategies and bioinformatic approaches. However, a comprehensive analysis of the effective secretome profile of Phytophthora is still lacking. Here, we report the first large-scale profiling of P. plurivora secretome using a shotgun LC-MS/MS strategy. To gain insight on the molecular signals underlying the cross-talk between plant pathogenic oomycetes and their host plants, we also investigate the quantitative changes of secreted protein following interaction of P. plurivora with the root exudate of Fagus sylvatica which is highly susceptible to the root pathogen. We show that besides known effectors, the expression and/or secretion levels of cell-wall-degrading enzymes were altered following the interaction with the host plant root exudate. In addition, a characterization of the F. sylvatica root exudate was performed by NMR and amino acid analysis, allowing the identification of the main released low-molecular weight components, including organic acids and free amino acids. This study provides important insights for deciphering the extracellular network involved in the highly susceptible P. plurivora-F. sylvatica interaction.

Comparison of binder compositions in Pompeian wall painting styles from Insula Occidentalis.

BACKGROUND: Although the pigment composition of Pompeian wall paintings has been the object of several studies, a comprehensive characterization of paint binder components is still lacking. This work aimed investigated at a molecular level the binder composition differences among wall paintings belonging to different periods of Pompeii's history. Analytical investigations were performed on representative samples of the first, second, third, and fourth painting styles excavated from the house of Marcus Fabius Rufus (Insula Occidentalis). The application of sensitive experimental methodologies was complemented by historical knowledge to gain insight in painting techniques and materials used by Pompeian artists. RESULTS: Fourier transform infrared spectroscopy and Raman spectroscopy were used to investigate the organic components and pigments present in powders obtained from samples of the four painting styles. No proteinaceous components were detected in the samples with liquid chromatography-electrospray ionization-hybrid quadrupole/time-of-flight mass spectrometry. Liquid chromatography, gas chromatography with flame-ionization detection, and gas chromatography-mass spectrometry of polar and non-polar components extracted from powders were used to evaluate and compare the free amino acids, sugars, and fatty acids profiles. CONCLUSIONS: Pigments and natural products (lipids, gums and wheat flours) were the main components of all samples. This supports the hypothesis that artists likely used water tempera for Pompeian wall paintings. Graphical AbstractScheme of the multi-analytical approach followed to compare Pompeian paint binders composition.Scheme of the multi-analytical approach followed to compare Pompeian paint binders composition.

Dramatic potentiation of the antiviral activity of HIV antibodies by cholesterol conjugation.

The broadly neutralizing antibodies HIV 2F5 and 4E10, which bind to overlapping epitopes in the membrane-proximal external region of the fusion protein gp41, have been proposed to use a two-step mechanism for neutralization; first, they bind and preconcentrate at the viral membrane through their long, hydrophobic CDRH3 loops, and second, they form a high affinity complex with the protein epitope. Accordingly, mutagenesis of the CDRH3 can abolish their neutralizing activity, with no change in the affinity for the peptide epitope. We show here that we can mimic this mechanism by conjugating a cholesterol group outside of the paratope of an antibody. Cholesterol-conjugated antibodies bind to lipid raft domains on the membrane, and because of this enrichment, they show increased antiviral potency. In particular, we find that cholesterol conjugation (i) rescues the antiviral activity of CDRH3-mutated 2F5, (ii) increases the antiviral activity of WT 2F5, (iii) potentiates the non-membrane-binding HIV antibody D5 10-100-fold (depending on the virus strain), and (iv) increases synergy between 2F5 and D5. Conjugation can be made at several positions, including variable and constant domains. Cholesterol conjugation therefore appears to be a general strategy to boost the potency of antiviral antibodies, and, because membrane affinity is engineered outside of the antibody paratope, it can complement affinity maturation strategies.

Anti-amyloidogenic property of human gastrokine 1.

Gastrokine 1 (GKN1) is a stomach-specific protein expressed in normal gastric tissue but absent in gastric cancer. GKN1 plays a major role in maintaining gastric mucosa integrity and is characterized by the presence of a BRICHOS domain consisting of about 100 amino acids also found in several unrelated proteins associated with major human diseases like BRI2, related to familial British and Danish dementia and surfactant protein C (SP-C), associated with respiratory distress syndrome. It was reported that recombinant BRICHOS domains from BRI2 and SP-C precursor (proSP-C) prevent fibrils formation of amyloid-beta peptide (Aß), that is the major component of extracellular amyloid deposits in Alzheimer's disease. Here we investigated on the interaction between human recombinant GKN1 (rGKN1) and Aß peptide (1-40) that derives from the partial hydrolysis of the amyloid precursor protein (APP). GKN1 prevented amyloid aggregation and fibrils formation by inhibiting Aß(1-40) polymerization, as evaluated by SDS-PAGE, thioflavin-T binding assay and gel filtration experiments. Mass spectrometry showed the formation of a prevailing 1:1 complex between GKN1 and Aß(1-40). SPR analysis of GKN1/Aß interaction led to calculate a dissociation constant (KD) of 34 µM. Besides its interaction with Aß(1-40), GKN1 showed also to interact with APP as evaluated by confocal microscopy and Ni-NTA pull-down. Data strongly suggest that GKN1 has anti-amyloidogenic properties thus functioning as a chaperone directed against unfolded segments and with the ability to recognize amyloidogenic polypeptides and prevent their aggregation.

The cold-adapted γ-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas haloplanktis.

A recombinant γ-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas haloplanktis (rPhGshA II) was produced and characterised. This enzyme catalyses the first step of glutathione biosynthesis by forming γ-glutamyl-cysteine from glutamate and cysteine in an ATP-dependent reaction. The other ATP-dependent enzyme, glutathione synthetase (rPhGshB), involved in the second step of the biosynthesis, was already characterised. rPhGshA II is a monomer of 58 kDa and its activity was characterised through a direct radioisotopic method, measuring the rate of ATP hydrolysis. The enzyme was active even at cold temperatures in a moderately alkaline buffer containing a high concentration of Mg(++); 2-aminobutyrate could replace cysteine, although a lower activity was detected. The reaction rate of rPhGshA II at 15 °C was higher than that reported for rPhGshB, thus suggesting that formation of γ-glutamyl-cysteine was not the rate limiting step of glutathione biosynthesis in P. haloplanktis. rPhGshA II had different affinities for its substrates, as evaluated on the basis of the KM values for ATP (0.093 mM), glutamate (2.8 mM) and cysteine (0.050 mM). Reduced glutathione acted as an inhibitor of rPhGshA II, probably through the binding to an enzyme pocket different from the active site. Also the oxidised form of glutathione inhibited the enzyme with a more complex inhibition profile, due to the complete mono-glutathionylation of rPhGshA II on Cys 386, as proved by mass spectrometry data. When compared to rPhGshB, rPhGshA II possessed more typical features of a psychrophilic enzyme, as it was endowed with lower thermodependence and higher heat sensitivity. In conclusion, this work extends the knowledge on glutathione biosynthesis in the first cold-adapted source; however, another possible redundant γ-glutamyl-cysteine ligase (PhGshA I), not yet characterised, could participate in the biosynthesis of this cellular thiol in P. haloplanktis.

Purification and characterization of novel cationic peroxidases from Asparagus acutifolius L. with biotechnological applications.

Four novel basic peroxidases, named AaP-1, AaP-2, AaP-3, and AaP-4, were purified from Asparagus acutifolius L. seeds by cation-exchange and gel filtration chromatographies. The four proteins showed a similar electrophoretic mobility of 46 kDa while, by MALDI-TOF MS, different Mr values of 42758.3, 41586.9, 42796.3, and 41595.5 were determined for AaP-1, AaP-2, AaP-3, and AaP-4, respectively. N-terminal sequences of AaPs 1-4 up to residue 20 showed a high percentage of identity with the peroxidase from Glycine max. In addition, AaP-1, AaP-2, AaP-3, and AaP-4 were found to be glycoproteins, containing 21.75, 22.27, 25.62, and 18.31 % of carbohydrates, respectively. Peptide mapping and MALDI-TOF MS analysis of AaPs 1-4 showed that the structural differences between AaP-1 and AaP-2 and AaP-3 and AaPs-4 were mainly due to their glycan content. We also demonstrate that AaPs were able to remove phenolic compounds from olive oil mill wastewaters with a higher catalytic efficiency with respect to horseradish peroxidase, thus representing candidate enzymes for potential biotechnological applications in the environmental field.

Saliva from obese individuals suppresses the release of aroma compounds from wine.

BACKGROUND: Recent evidence suggests that a lower extent of the retronasal aroma release correspond to a higher amount of ad libitum food intake. This has been regarded as one of the bases of behavioral choices towards food consumption in obese people. In this pilot study we investigated the hypothesis that saliva from obese individuals could be responsible for an alteration of the retro-nasal aroma release. We tested this hypothesis in vitro, by comparing the release of volatiles from a liquid food matrix (wine) after its interaction with saliva from 28 obese (O) and 28 normal-weight (N) individuals. METHODS AND FINDINGS: Amplicon sequencing of the 16S rRNA V4 region indicated that Firmicutes and Actinobacteria were more abundant in O, while Proteobacteria and Fusobacteria dominated in N. Streptococcaceae were significantly more abundant in the O subjects and constituted 34% and 19% on average of the saliva microbiota of O and N subjects, respectively. The Total Antioxidant Capacity was higher in O vs N saliva samples. A model mouth system was used to test whether the in-mouth wine aroma release differs after the interaction with O or N saliva. In O samples, a 18% to 60% significant decrease in the mean concentration of wine volatiles was detected as a result of interaction with saliva, compared with N. This suppression was linked to biochemical differences in O and N saliva composition, which include protein content. CONCLUSION: Microbiological and biochemical differences were found in O vs N saliva samples. An impaired retronasal aroma release from white wine was detected in vitro and linked to compositional differences between saliva from obese and normal-weight subjects. Additional in vivo investigations on diverse food matrices could contribute to understanding whether a lower olfactory stimulation due to saliva composition can be a co-factor in the development/maintenance of obesity.

An integrated approach based on multiplexed protein array and iTRAQ labeling for in-depth identification of pathways associated to IVF outcome.

The emergence of high-throughput protein quantification methodologies has enabled the comprehensive characterization by longitudinal and cross-sectional studies of biological fluids under physiological and pathological conditions. In particular, the simultaneous investigation of cytokines and growth factors signaling pathways and their associated downstream effectors by integrated multiplexed approaches offers a powerful strategy to gain insights into biological networks and processes in living systems. A growing body of research indicates that bioactive molecules of human reproductive fluids, including human follicular fluid (hFF), may affect oocyte quality, fertilization and embryo development, thus potentially influencing the physiopathology of pregnancy-related conditions. In this work, an iTRAQ labeling strategy has been complemented with a multiplexed protein array approach to analyze hFFs with the aim to investigate biological processes and pathways related to in vitro fertilization (IVF) outcome. The iTRAQ labeling strategy lead to the quantification of 89 proteins, 30 of which were differentially expressed in hFFs with successful compared to unsuccessful IVF outcome. The targeted study, based on multiplexed antibody protein arrays, allowed the simultaneous quantification of 27 low abundance proteins, including growth factors, chemokines and cytokines endowed with pro- and anti-inflammatory activity. A significant number of differentially regulated proteins were involved in biological functions related to blood coagulation, acute phase response signaling and complement system. Overall, the present results provide an integrated overview of protein changes in hFFs associated to IVF outcome, thus improving current knowledge in reproductive medicine and fertility research.

Spectroscopic identification and anti-biofilm properties of polar metabolites from the medicinal plant Helichrysum italicum against Pseudomonas aeruginosa.

Two new acylated styrylpyrones, one 5-methoxy-1(3H)-isobenzofuranone glucoside and a hydroxymethyl-orcinol derivative, along with sixteen known aromatic metabolites, including lignans, quinic acid derivatives low-molecular weight phenol glucosides, have been isolated from the methanol extract of Helichrysum italicum, a medicinal plant typical of the Mediterranean vegetation. The structures of these compounds have been elucidated on the basis of extensive 2D-NMR spectroscopic analyses, including COSY, TOCSY, HSQC, CIGAR-HMBC, H2BC and HSQC-TOCSY, along with Q-TOF HRMS(2) analysis. Selected compounds were evaluated for their anti-biofilm properties against Pseudomonas aeruginosa.