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.

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.

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.

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.

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.

Secretome profiling of differentiated neural mes-c-myc A1 cell line endowed with stem cell properties.

Neural stem cell proliferation and differentiation play a crucial role in the formation and wiring of neuronal connections forming neuronal circuits. During neural tissues development, a large diversity of neuronal phenotypes is produced from neural precursor cells. In recent years, the cellular and molecular mechanisms by which specific types of neurons are generated have been explored with the aim to elucidate the complex events leading to the generation of different phenotypes via distinctive developmental programs that control self-renewal, differentiation, and plasticity. The extracellular environment is thought to provide instructive influences that actively induce the production of specific neuronal phenotypes. In this work, the secretome profiling of differentiated neural mes-c-myc A1 (A1) cell line endowed with stem cell properties was analyzed by applying a shotgun LC-MS/MS approach. The results provide a list of secreted molecules with potential relevance for the functional and biological features characterizing the A1 neuronal phenotype. Proteins involved in biological processes closely related to nervous system development including neurites growth, differentiation of neurons and axonogenesis were identified. Among them, proteins belonging to extracellular matrix and cell-adhesion complexes as well as soluble factors with well established neurotrophic properties were detected. The presented work provides the basis to clarify the complex extracellular protein networks implicated in neuronal differentiation and in the acquisition of the neuronal phenotype. This article is part of a Special Issue entitled: An Updated Secretome.

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.

Synthesis and biological properties of caffeic acid-PNA dimers containing guanine.

Caffeic acid (CA; 3,4-dihydroxycinnamic acid) is endowed with high antioxidant activity. CA derivatives (such as amides) have gained a lot of attention due to their antioxidative, antitumor and antimicrobial properties as well as stable characteristics. Caffeoyl-peptide derivatives showed different antioxidant activity depending on the type and the sequence of amino acid used. For these reasons, we decided to combine CA with Peptide Nucleic Acid (PNA) to test whether the new PNA-CA amide derivatives would result in an improvement or gain of CA's biological (i.e., antioxidant, cytotoxic, cytoprotective) properties. We performed the synthesis and characterization of seven dimer conjugates with various combinations of nucleic acid bases and focused NMR studies on the model compound ga-CA dimer. We demonstrate that PNA dimers containing guanine conjugated to CA exhibited different biological activities depending on composition and sequence of the nucleobases. The dimer ag-CA protected HepG2, SK-B-NE(2), and C6 cells from a cytotoxic dose of hydrogen peroxide (H2O2).

Structural and enzymatic properties of an in vivo proteolytic form of PD-S2, type 1 ribosome-inactivating protein from seeds of Phytolacca dioica L.

PD-S2, type 1 ribosome-inactivating protein from Phytolacca dioica L. seeds, is an N-ß-glycosidase likely involved in plant defence. In this work, we purified and characterized an in vivo proteolytic form of PD-S2, named cutPD-S2. Spectroscopic characterization of cutPD-S2 showed that the proteolytic cleavage between Asn195 and Arg196 does not alter the protein fold, but significantly affects its thermal stability. Most importantly, the proteolytic cleavage induces a 370-fold decrease of PD-S2 capacity of inhibiting in vitro protein biosynthesis. Our data catch the turning point from a typical role of PD-S2 as a defence protein to that of supplier of essential amino acids during seedling development.

Polar and non-polar organic binder characterization in Pompeian wall paintings: comparison to a simulated painting mimicking an "a secco" technique.

The use of Fourier transform infrared spectromicroscopy and mass spectrometry (MS) allowed us to characterize the composition of polar and non-polar binders present in sporadic wall paint fragments taken from Pompeii's archaeological excavation. The analyses of the polar and non-polar binder components extracted from paint powder layer showed the presence of amino acids, sugars, and fatty acids but the absence of proteinaceous material. These results are consistent with a water tempera painting mixture composed of pigments, flours, gums, and oils and are in agreement with those obtained from a simulated wall paint sample made for mimicking an ancient "a secco" technique. Notably, for the first time, we report the capability to discriminate by tandem MS the presence of free amino acids in the paint layer.

Characterization of pigments and ligands in a wall painting fragment from Liternum archaeological park (Italy).

Spectroscopic and MS techniques were used to characterize the pigments and the composition of polar and nonpolar binders of a stray wall painting fragment from Liternum (Italy) archaeological excavation. X-ray fluorescence and diffraction analysis of the decorations indicated mainly the presence of calcite, quartz, hematite, cinnabar, and cuprorivaite. Infrared spectroscopy, GC coupled to flame-ionization detector, and MS analysis of the polar and nonpolar components extracted from paint layers from three different color regions revealed the presence of free amino acids, sugars, and fatty acids. Interestingly, LC-MS shotgun analysis of the red painting region showed the presence of αS1-casein of buffalo origin. Compared to our previous results from Pompeii's wall paintings, even though the Liternum painting mixture contained also binders of animal origin, the data strongly suggest that in both cases a tempera painting technique was utilized.

Temporal proteomic profiling of embryonic stem cell secretome during cardiac and neural differentiation.

During recent years, increased efforts have focused on elucidating the pluripotency and self-renewal of stem cells. Differentiation towards the different lineages has attracted significant attention given the potential use of stem cells in regenerative medicine. Embryonic stem cell differentiation is a complex process coordinated by strictly regulated extracellular signals that act in an autocrine and/or paracrine manner. Through secreted molecules, stem cells affect local niche biology and influence the cross-talking with the surrounding tissues. Emerging evidence supports the hypothesis that fundamental cell functions, including proliferation and differentiation, are strictly regulated by the complex set of molecules secreted from cells. The understanding of this molecular language could largely increase our knowledge on pathways regulating stem cell differentiation. Here, we have used a proteomics platform to investigate the profile of proteins secreted during differentiation of murine embryonic stem cells. We have followed the dynamics of protein secretion by comparing the secretomes at different time points of murine embryonic stem cell cardiac and neural differentiation. In addition to previously reported molecules, we have identified many secreted proteins not described so far as released from embryonic stem cells nor shown to be differentially released during the process of cardiomyogenesis and neurogenesis.

Proteomic characterization of early changes induced by triiodothyronine in rat liver.

High doses of T3 are mitogenic in liver, causing hyperplasia that has numerous differences from the compensatory regeneration induced by partial hepatectomy (PH). T3 binds to the thyroid hormone receptor (TR), which directly regulates transcription, while PH acts indirectly through signal transduction pathways. We therefore carried out a proteomic analysis to compare early effects of the two treatments. Transcriptome analysis by DNA microarray also confirmed the observed proteomic changes, demonstrating that they were caused by transcriptional regulation. Among the differentially expressed proteins, many are directly or indirectly involved in energy metabolism and response to oxidative stress. Several enzymes of lipid metabolism (e.g., Acaa2, Acads, Hadh, and Echs1) were differentially regulated by T3. In addition, altered expression levels of several mitochondrial proteins (e.g., Hspa9, Atp5b, Cps1, Glud1, Aldh2, Ak2, Acads) demonstrated the known increase of mitochondrial biogenesis mediated by T3. The present results provide insights in changes in metabolic balance occurring following T3-stimulation and define a basis for dissecting the molecular pathways of hepatocyte hyperplasia.

WCI, a novel wheat chymotrypsin inhibitor: purification, primary structure, inhibitory properties and heterologous expression.

A novel chymotrypsin inhibitor, detected in the endosperm of Triticum aestivum, was purified and characterized with respect to the main physical-chemical properties. On the basis of its specificity, this inhibitor was named WCI (wheat chymotrypsin inhibitor). WCI is a monomeric neutral protein made up of 119 residues and molecular mass value of 12,933.40 Da. Automated sequence and mass spectrometry analyses, carried out on several samples of purified inhibitor, evidenced an intrinsic molecular heterogeneity due to the presence of the isoform [des-(Thr)WCI], accounting for about 40% of the total sample. In vitro, WCI acted as a strong inhibitor of bovine pancreatic chymotrypsin as well as of chymotryptic-like activities isolated from the midgut of two phytophagous insects, Helicoverpa armigera (Hüb.) and Tenebrio molitor L., respectively. No inhibitory activities were detected against bacterial subtilisins, bovine pancreatic trypsin, porcine pancreatic elastase or human leukocyte elastase. The primary structure of WCI was significantly similar (45.7-89.1%) to those of several proteins belonging to the cereal trypsin/α-amylase inhibitor super-family and showed the typical sequence motif of this crowed protein group. The cDNA of the inhibitor (wci-cDNA) was isolated from wheat immature caryopses and employed to obtain a recombinant product in E. coli. Experimental evidences indicated that the recombinant inhibitor was localized in the inclusion bodies from which it was recovered as soluble and partially active protein by applying an appropriate refolding procedure. WCI reactive site localization, as well as its inhibitory specificity, was investigated by molecular modeling approach.

Proteomic analysis of human U937 cell line activation mediated by Haemophilus influenzae type b P2 porin and its surface-exposed loop 7.

The virulence of Haemophilus influenzae type b (Hib) has been attributed to a variety of potential factors associated with its cell surface, including lipopolysaccharides (LPS) and major outer membrane proteins (OMPs). P2 porin, one of the best-characterized porins in terms of its functional characteristics, is the most abundant OMP in Hib and has also been shown to possess proinflammatory activity. To characterize the role played by bacterial surface components in disease onset and development, the proteomic profiling of human U937 cell line activated by H. influenzae type b P2 porin and its most active surface-exposed loop (L7) was performed by means of two-dimensional electrophoresis and mass spectrometry. The study provided a list of candidate proteins with potential relevance in the host immune and inflammatory response. Most of the differentially expressed proteins are involved in metabolic processes, remodelling of cytoskeleton, stress response and signal transduction pathways. The results constitute the basis for dissecting signal transduction cascades activated by P2 stimulation and gain insights into the molecular events involved in the modulation of pathogen-host cell interactions.

Expression of the brain transcription factor OTX1 occurs in a subset of normal germinal-center B cells and in aggressive Non-Hodgkin Lymphoma.

The roles in brain development. Previous studies have shown the association between OTX2 and OTX1 with anaplastic and desmoplastic medulloblastomas, respectively. Here, we investigated OTX1 and OTX2 expression in Non-Hodgkin Lymphoma (NHL) and multiple myeloma. A combination of semiquantitative RT-PCR, Western blot, and immunohistochemical analyses was used to measure OTX1 and OTX2 levels in normal lymphoid tissues and in 184 tumor specimens representative of various forms of NHL and multiple myeloma. OTX1 expression was activated in 94% of diffuse large B-cell lymphomas, in all Burkitt lymphomas, and in 90% of high-grade follicular lymphomas. OTX1 was undetectable in precursor-B lymphoblastic lymphoma, chronic lymphocytic leukemia, and in most marginal zone and mantle cell lymphomas and multiple myeloma. OTX2 was undetectable in all analyzed malignancies. Analysis of OTX1 expression in normal lymphoid tissues identified a subset of resting germinal center (GC) B cells lacking PAX5 and BCL6 and expressing cytoplasmic IgG and syndecan. About 50% of OTX1(+) GC B cells co-expressed CD10 and CD20. This study identifies OTX1 as a molecular marker for high-grade GC-derived NHL and suggests an involvement of this transcription factor in B-cell lymphomagenesis. Furthermore, OTX1 expression in a subset of normal GC B cells carrying plasma cell markers suggests its possible contribution to terminal B-cell differentiation.

Quantification of thyrotropin-releasing hormone by liquid chromatography-electrospray mass spectrometry.

Thyrotropin-releasing hormone (TRH) is involved in a wide range of biological responses. It has a central role in the endocrine system and regulates several neurobiological activities. In the present study, a rapid, sensitive and selective liquid chromatography-mass spectrometry method for the identification and quantification of TRH has been developed. The methodology takes advantage of the specificity of the selected-ion monitoring acquisition mode with a limit of detection of 1 fmol. Furthermore, the MS/MS fragmentation pattern of TRH has been investigated to develop a selected reaction monitoring (SRM) method that allows the detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid-histidine. The method has been tested on rat hypothalami to evaluate its suitability for the detection within very complex biological samples.

Improved procedure for protein binder analysis in mural painting by LC-ESI/Q-q-TOF mass spectrometry: detection of different milk species by casein proteotypic peptides.

Diagnostic techniques applied to the field of cultural heritage represent a very important aspect of scientific investigation. Recently, proteomic approaches based on mass spectrometry coupled with traditional spectroscopic methods have been used for painting analysis, generating promising results for binder's protein identification. In the present work, an improved procedure based on LC-ESI/Q-q-TOF tandem mass spectrometry for the identification of protein binders has been developed for the molecular characterization of samples from an early-twentieth-century mural painting from the St. Dimitar Cathedral in Vidin, Bulgaria. The proteomic investigation has led to the identification of both egg white and egg yolk proteins, according to traditional old recipes for tempera paintings. In addition, beyond the egg components, the presence of caseins was also revealed, thus suggesting the use of milk as binding medium, fixative or stabilising agent. Furthermore, for the first time, the capability to discriminate the milk origin on the basis of alpha casein proteotypic peptides is reported, that are diagnostic for a given species, thus opening interesting perspectives in art and archaeological fields.

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.