Introduction to the Special Issue Dedicated to Extracellular Vesicles and Nanoparticles, Part 1.

The existence of extracellular vesicles [EVs] has been known for more than eighty years, [...].

Possible Role of Extracellular Vesicles in Hepatotoxicity of Acetaminophen.

We examined proteomic profiles of rat liver extracellular vesicles (EVs) shed following treatment with a sub-toxic dose (500 mg/kg) of the pain reliever drug, acetaminophen (APAP). EVs representing the entire complement of hepatic cells were isolated after perfusion of the intact liver and analyzed with LC-MS/MS. The investigation was focused on revealing the function and cellular origin of identified EVs proteins shed by different parenchymal and non-parenchymal liver cells and their possible role in an early response of this organ to a toxic environment. Comparison of EV proteomic profiles from control and APAP-treated animals revealed significant differences. Alpha-1-macroglobulin and members of the cytochrome P450 superfamily were highly abundant proteins in EVs shed by the normal liver. In contrast, proteins like aminopeptidase N, metalloreductase STEAP4, different surface antigens like CD14 and CD45, and most members of the annexin family were detected only in EVs that were shed by livers of APAP-treated animals. In EVs from treated livers, there was almost a complete disappearance of members of the cytochrome P450 superfamily and a major decrease in other enzymes involved in the detoxification of xenobiotics. Additionally, there were proteins that predominated in non-parenchymal liver cells and in the extracellular matrix, like fibronectin, receptor-type tyrosine-protein phosphatase C, and endothelial type gp91. These differences indicate that even treatment with a sub-toxic concentration of APAP initiates dramatic perturbation in the function of this vital organ.

Changes in the proteome of extracellular vesicles shed by rat liver after subtoxic exposure to acetaminophen.

To identify changes in extracellular vesicles (EVs) secreted by the liver following drug-induced liver injury (DILI), rats were treated with a subtoxic dose (500 mg/kg) of the analgesic drug, acetaminophen (APAP). EVs were collected by liver perfusion of sham and APAP-treated rats. Changes in EVs morphology were examined by transmission electron microscopic analysis of negatively stained vesicles. Results from morphometric analysis of EVs revealed striking differences in their size and distribution. Proteome composition of EVs collected by liver perfusion was determined by mass spectrometry using methods of sample preparation that enabled better detection of both highly hydrophobic proteins and proteins with complex post-translational modifications. The collection of EVs after liver perfusion is an approach that enables the isolation of EVs shed not only by isolated hepatocytes, but also by the entire complement of hepatic cells. EVs derived after DILI had a lower content of alpha-1-macroglobulin, ferritin, and members of cytochrome 450 family. Fibronectin, aminopeptidase N, metalloreductase STEAP4, integrin beta, and members of the annexin family were detected only in APAP-treated samples of EVs. These results show that the present approach can provide valuable insights into the response of the liver following drug-induced liver injury.

Increased yield of enzymatic synthesis by chromatographic selection of different N-glycoforms of yeast invertase.

Invertases are glycosidases applied for synthesis of alkyl glycosides that are important and effective surfactants. Stability of invertases in the environment with increased content of organic solvent is crucial for increase of productivity of glycosidases. Their stability is significantly influenced by N-glycosylation. However, yeast N-glycosylation pathways may synthesize plethora of N-glycan structures. A total natural crude mixture of invertase glycoforms (EINV) extracted from Saccharomyces cerevisiae was subfractionated by anion-exchange chromatography on industrial monolithic supports to obtain different glycoforms (EINV1-EINV3). Separated glycoforms exhibited different stabilities in water-alcohol solutions that are in direct correlation with the amount of phosphate bound to N-glycans. Observed differences in stability of different invertase glycoforms were used to improve productivity of methyl ß-d-fructofuranoside (MF) synthesis. The efficiency and yield of MF synthesis were improved more than 50% when the most stabile glycoform bearing the lowest amount of phosphorylated N-glycans is selected and utilized. These data underline the importance of analysis of glycan structures attached to glycoproteins, demonstrate different impact of N-glycans on the surface charge and enzyme stability in regard to particular reaction environment, and provide a platform for improvement of yield of industrial enzymatic synthesis by chromatographic selection of glycoforms on monolithic supports.

Biofilm formation and extracellular microvesicles-The way of foodborne pathogens toward resistance.

Almost all known foodborne pathogens are able to form biofilms as one of the strategies for survival under harsh living conditions, to ward off the inhibition and the disinfection during food production, transport and storage, as well as during cleaning and sanitation of corresponding facilities. Biofilms are communities where microbial cells live under constant intracellular interaction and communication. Members of the biofilm community are embedded into extracellular matrix that contains polysaccharides, DNA, lipids, proteins, and small molecules that protect microorganisms and enable their intercellular communication under stress conditions. Membrane vesicles (MVs) are produced by both Gram positive and Gram negative bacteria. These lipid membrane-enveloped nanoparticles play an important role in biofilm genesis and in communication between different biofilm members. Furthermore, MVs are involved in other important steps of bacterial life like cell wall modeling, cellular division, and intercellular communication. They also carry toxins and virulence factors, as well as nucleic acids and different metabolites, and play a key role in host infections. After entering host cells, MVs can start many pathologic processes and cause serious harm and cell death. Prevention and inhibition of both biofilm formation and shedding of MVs by foodborne pathogens has a very important role in food production, storage, and food safety in general. Better knowledge of biofilm formation and maintaining, as well as the role of microbial vesicles in this process and in the process of host cells' infection is essential for food safety and prevention of both food spoilage and host infection.

Glycosylation and metastases.

The change of cellular glycosylation is one of the key events in malignant transformation and neoplastic progression, and tumor-related glycosylation alterations are promising targets in both tumor diagnosis and therapy. Both malignant transformation and neoplastic progression are the consequence of gene expression alterations and alterations in protein expression. Micro environmental factors such as extracellular matrix (ECM) also play an important role in their growth and metastasis. Tumor-associated glycans are important biomarker candidates for cancer diagnosis and prognosis, and analytical methods for their detection were developed recently. Glycoproteomics that use mass spectrometry for identification of cancer antigens and structural analysis of glycans play a key role in the investigation of changes of glycosylation during malignant transformation and tumor development and metastasis. Deep understanding of glycan remodeling in cancer and the role of glycosyltransferases that are involved in this process will require a detailed profiling of glycosylation patterns of tumor cells, and corresponding analytical methods for their detection were developed.

Sample preparation in foodomic analyses.

Representative sampling and adequate sample preparation are key factors for successful performance of further steps in foodomic analyses, as well as for correct data interpretation. Incorrect sampling and improper sample preparation can be sources of severe bias in foodomic analyses. It is well known that both wrong sampling and sample treatment cannot be corrected anymore. These, in the past frequently neglected facts, are now taken into consideration, and the progress in sampling and sample preparation in foodomics is reviewed here. We report the use of highly sophisticated instruments for both high-performance and high-throughput analyses, as well as miniaturization and the use of laboratory robotics in metabolomics, proteomics, peptidomics, and genomics.

Soft agar-based selection of spontaneously transformed rat prostate epithelial cells with highly tumorigenic characteristics.

The critical molecular and cellular mechanisms involved in the development and progression of prostate cancer remain elusive. In this report, we demonstrate that normal rat prostate epithelial cells (PEC) undergo spontaneous transformation at high passage (p > 85) evidenced by the acquisition of anchorage independent growth when plated on soft agar and tumorigenicity when injected into immunodeficient mice. In addition, we also report the discovery of a minor subpopulation of spontaneously transformed PEC derived from high passage PEC with the ability to migrate through a layer of 1% agar and form expanding colonies on the underlying plastic substratum. Comparison of these soft agar invasive (SAI) cells with low (p < 35), mid (p36-84) and high passage (p > 85) PEC identified marked differences in cell morphology, proliferation and motility. The SAI subpopulation was more tumorigenic than the high passage anchorage independent cultures from which they were isolated, as manifested by a decreased latency period and an increase in the size of tumors arising in immunodeficient mice. In contrast, low and mid passage cells were unable to grow on soft agar and failed to form tumors when injected into immunodeficient mice. Screening with antibody-based signaling arrays identified several differences in the altered expression levels of signaling proteins between SAI-derived cells and low or high passage PEC, including the up-regulation of EGFR and MAPK-related signaling pathways in SAI-selected cells. In summary, these studies suggest that the SAI assay selects for a novel, highly tumorigenic subpopulation of transformed cells that may represent an early step in the progression of slow growing prostatic carcinomas into more rapidly growing and aggressive tumors.

IgG and IgM glycosylation patterns in patients undergoing image-guided tumor ablation.

BACKGROUND: Image-guided tumor ablation is a technique whereby needle-like applicators are placed directly into solid tumors under guidance typically with computed tomography or ultrasound. Changes in IgG and IgM antibody glycosylation were studied during ablation-induced immune response to cancer, and the use of glycosylation as a biomarker for diagnosis, prognosis and disease treatment was examined. METHODS: Plasma from 27 tumor patients was collected immediately before, after and for 6 months following ablation. IgG and IgM antibodies were isolated by use high-throughput chromatography, and analyzed by hydrophilic liquid chromatography. Thorough identification of glycan structures in each chromatography peak was performed by nano-liquid chromatography electrospray ionization mass spectrometry. RESULTS: Although antibody glycosylation was found to vary with cancer type, discernable patterns of change based on the successful treatment of tumors by ablation were not identified. One patient with renal clear cell carcinoma and poor disease outcome had unexpectedly high amount of oligomannose IgG glycans during the whole period of monitoring. In contrast, IgM antibodies did not follow the same pattern. CONCLUSIONS: These findings suggest that glycosylation patterns are indicative of an immune system that is unable to prevent different types of cancer, rather than products of the immunostimulatory response to the ablation of tumor itself. Analyses of the outcome effect suggested that IgG glycosylation and IgM glycosylation are not associated with tumor ablation. GENERAL SIGNIFICANCE: Present work opens a new way for parallel determination of glycosylation changes of both IgG and IgM antibodies by use of high-throughput methods, and their future use as biomarkers for disease diagnosis and prognosis. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

Polymethacrylate-based monoliths as stationary phases for separation of biopolymers and immobilization of enzymes.

The experiences in the production and application of polymethacrylate-based monolithic supports, since their development almost thirty years ago, are presented. The main driving force for the development of new chromatographic supports was the necessity for the isolation and separation of physiologically active biopolymers and their use for therapeutic purposes. For this sake, a development of a method for fast separation, preventing denaturation and preserving their biological activity was necessary. Development of polysaccharide-based supports, followed by the introduction of polymer-based chromatographic media, is shortly described. This development was followed by the advances in monolithic media that are now used for both large- and small-scale separation of biopolymers and nanoparticles. Finally, a short overview is given about the applications of monoliths for sample displacement chromatography, resulting in isolation of physiologically active biomolecules, such as proteins, protein complexes, and nucleic acid, as well as high-throughput sample preparation for proteomic investigations.

Affinity chromatography on monolithic supports for simultaneous and high-throughput isolation of immunoglobulins from human serum.

Posttranslational modifications of immunoglobulins have been a topic of great interest and have been repeatedly reported as a major factor in disease pathology. Cost-effective, reproducible, and high-throughput (HTP) isolation of immunoglobulins from human serum is vital for studying the changes in protein structure and the following understanding of disease development. Although there are many methods for the isolation of specific immunoglobulin classes, only a few of them are applicable for isolation of all subtypes and variants. Here, we present the development of a scheme for fast and simultaneous affinity purification of α (A), γ (G), and µ (M) immunoglobulins from human serum through affinity monolith chromatography. Affinity-based monolithic columns with immobilized protein A, G, or L were used for antibody isolation. Monolithic stationary phases have a high surface accessibility of binding sites, large flow-through channels, and can be operated at high flow rates, making them the ideal supports for HTP isolation of biopolymers. The presented method can be used for HTP screening of human serum in order to simultaneously isolate all three above-mentioned immunoglobulins and determine their concentration and changes in their glycosylation pattern as potential prognostic and diagnostic disease biomarkers.

Secretome analysis of an osteogenic prostate tumor identifies complex signaling networks mediating cross-talk of cancer and stromal cells within the tumor microenvironment.

A distinct feature of human prostate cancer (PCa) is the development of osteoblastic (bone-forming) bone metastases. Metastatic growth in the bone is supported by factors secreted by PCa cells that activate signaling networks in the tumor microenvironment that augment tumor growth. To better understand these signaling networks and identify potential targets for therapy of bone metastases, we characterized the secretome of a patient-derived xenograft, MDA-PCa-118b (PCa-118b), generated from osteoblastic bone lesion. PCa-118b induces osteoblastic tumors when implanted either in mouse femurs or subcutaneously. To study signaling molecules critical to these unique tumor/microenvironment-mediated events, we performed mass spectrometry on conditioned media of isolated PCa-118b tumor cells, and identified 26 secretory proteins, such as TGF-ß2, GDF15, FGF3, FGF19, CXCL1, galectins, and ß2-microglobulin, which represent both novel and previously published secreted proteins. RT-PCR using human versus mouse-specific primers showed that TGFß2, GDF15, FGF3, FGF19, and CXCL1 were secreted from PCa-118b cells. TGFß2, GDF15, FGF3, and FGF19 function as both autocrine and paracrine factors on tumor cells and stromal cells, that is, endothelial cells and osteoblasts. In contrast, CXCL1 functions as a paracrine factor through the CXCR2 receptor expressed on endothelial cells and osteoblasts. Thus, our study reveals a complex PCa bone metastasis secretome with paracrine and autocrine signaling functions that mediate cross-talk among multiple cell types within the tumor microenvironment.

Foodomics and Food Safety: Where We Are.

The power of foodomics as a discipline that is now broadly used for quality assurance of food products and adulteration identification, as well as for determining the safety of food, is presented. Concerning sample preparation and application, maintenance of highly sophisticated instruments for both high-performance and high-throughput techniques, and analysis and data interpretation, special attention has to be paid to the development of skilled analysts. The obtained data shall be integrated under a strong bioinformatics environment. Modern mass spectrometry is an extremely powerful analytical tool since it can provide direct qualitative and quantitative information about a molecule of interest from only a minute amount of sample. Quality of this information is influenced by the sample preparation procedure, the type of mass spectrometer used and the analyst's skills. Technical advances are bringing new instruments of increased sensitivity, resolution and speed to the market. Other methods presented here give additional information and can be used as complementary tools to mass spectrometry or for validation of obtained results. Genomics and transcriptomics, as well as affinity-based methods, still have a broad use in food analysis. Serious drawbacks of some of them, especially the affinity-based methods, are the cross-reactivity between similar molecules and the influence of complex food matrices. However, these techniques can be used for pre-screening in order to reduce the large number of samples. Great progress has been made in the application of bioinformatics in foodomics. These developments enabled processing of large amounts of generated data for both identification and quantification, and for corresponding modeling.

Proteomics profiling of keratocystic odontogenic tumours reveals AIDA as novel biomarker candidate.

BACKGROUND: Keratocystic odontogenic tumour (KCOT) is a benign, yet aggressive odontogenic tumour. Herein, proteome analysis of KCOT lesions in comparison with control patient-matched tissue unaffected by the disease and with inflammatory odontogenic cysts, namely radicular cysts is presented. METHODS: For the proteomics profiling, two complementary proteomics techniques MALDI-MS/MS and LC-ESI-MS/MS were employed. Potential candidate biomarkers were validated by immunohistochemistry. RESULTS: More than 43 proteins were found to be differentially expressed or up-regulated in KCOT lesions in comparison with patient-matched unaffected oral mucosa. These proteins bear important biological functions and are involved in cell proliferation, cytoskeletal re-organization, transcription, cellular motility and apoptosis. In particular, a number of differentially expressed proteins participate in autocrine regulation and signalization within JNK and p38 MAPK signalling pathways. CONCLUSIONS: Immunohistochemical validation of chosen putative biomarkers revealed axin interaction partner and dorsalization-antagonist (AIDA), known as a protein that blocks activation of JNK signalling pathway, as a differential biomarker for KCOT lesions on an independent cohort of KCOT tissue samples in comparison with most prevalent intra-oseal lesions inflammatory odontogenic cysts.

Mass spectrometry-based analysis of rat liver and hepatocellular carcinoma Morris hepatoma 7777 plasma membrane proteome.

The gel-based proteomic analysis of plasma membranes from rat liver and chemically induced, malignant hepatocellular carcinoma Morris hepatoma 7777 was systematically optimized to yield the maximum number of proteins containing transmembrane domains (TMDs). Incorporation of plasma membrane proteins into a polyacrylamide "tube gel" followed by in-gel digestion of "tube gel" pieces significantly improved detection by electrospray ionization-liquid chromatography-tandem mass spectrometry. Removal of less hydrophobic proteins by washing isolated plasma membranes with 0.1 M sodium carbonate enables detection of a higher number of hydrophobic proteins containing TMDs in both tissues. Subsequent treatment of plasma membranes by a proteolytic enzyme (trypsin) causes the loss of some of the proteins that are detected after washing with sodium carbonate, but it enables the detection of other hydrophobic proteins containing TMDs. Introduction of mass spectrometers with higher sensitivity, higher mass resolution and mass accuracy, and a faster scan rate significantly improved detection of membrane proteins, but the improved sample preparation is still useful and enables detection of additional hydrophobic proteins. Proteolytic predigestion of plasma membranes enables detection of additional hydrophobic proteins and better sequence coverage of TMD-containing proteins in plasma membranes from both tissues.

Mixed electrolytes in hydrophobic interaction chromatography.

An essential part of the modulation of protein-binding capacity in hydrophobic interaction chromatography is the buffer-salt system. Besides using "single" electrolytes, multicomponent electrolyte mixtures may be used as an additional tool. Both the protein solubility and the binding capacity depend on the position of a salt in the so-called Hofmeister series. Specific interactions are observed for an individual protein-salt combination. For salt mixtures, selectivity, recovery, and binding capacity do not behave like for the single salts that are positioned in between the two mixed components in the Hofmeister series, as the continuous correlation would suggest. Thus, finding strategies for mixed salts could potentially lead to improved capacities in hydrophobic interaction chromatography. Mixtures of ammonium sulfate, sodium citrate, sodium sulfate, sodium chloride, sodium acetate, and glycine were used to investigate the binding capacities for lysozyme and a monoclonal antibody on various hydrophobic resins. Resin capacity for two investigated proteins increases when mixtures consisting of a chaotropic and a kosmotropic salt are applied. It seems to be related to the rather basic isoelectric points of the proteins.

High throughput isolation and glycosylation analysis of IgG-variability and heritability of the IgG glycome in three isolated human populations.

All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously. Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individual's age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs.

Human-specific features and developmental dynamics of the brain N-glycome.

Comparative "omics" studies have revealed unique aspects of human neurobiology, yet an evolutionary perspective of the brain N-glycome is lacking. Here, we performed multi-regional characterization of rat, macaque, chimpanzee, and human brain N-glycomes using chromatography and mass spectrometry, then integrated these data with complementary glycotranscriptomic data. We found that in primates the brain N-glycome has evolved more rapidly than the underlying transcriptomic framework, providing a mechanism for generating additional diversity. We show that brain N-glycome evolution in hominids has been characterized by an increase in complexity and α(2-6)-linked N-acetylneuraminic acid along with human-specific cell-type expression of key glycogenes. Finally, by comparing the prenatal and adult human brain N-glycome, we identify region-specific neurodevelopmental pathways that lead to distinct spatial N-glycosylation profiles in the mature brain. One-Sentence Summary: Evolution of the human brain N-glycome has been marked by an increase in complexity and a shift in sialic acid linkage.

Human-specific features and developmental dynamics of the brain N-glycome.

Comparative "omics" studies have revealed unique aspects of human neurobiology, yet an evolutionary perspective of the brain N-glycome is lacking. We performed multiregional characterization of rat, macaque, chimpanzee, and human brain N-glycomes using chromatography and mass spectrometry and then integrated these data with complementary glycotranscriptomic data. We found that, in primates, the brain N-glycome has diverged more rapidly than the underlying transcriptomic framework, providing a means for rapidly generating additional interspecies diversity. Our data suggest that brain N-glycome evolution in hominids has been characterized by an overall increase in complexity coupled with a shift toward increased usage of α(2-6)-linked N-acetylneuraminic acid. Moreover, interspecies differences in the cell type expression pattern of key glycogenes were identified, including some human-specific differences, which may underpin this evolutionary divergence. Last, by comparing the prenatal and adult human brain N-glycomes, we uncovered region-specific neurodevelopmental pathways that lead to distinct spatial N-glycosylation profiles in the mature brain.