Optimization of assay conditions to quantify ECOD activity in vivo in individual Daphnia magna. Assay performance evaluation with model CYP 450 inducers/inhibitors.

Screening the activity of the cytochrome P450 (CYP450) mixed function oxidase system in aquatic invertebrates received seldom applications in ecotoxicology due to low baseline enzymatic activities characteristic for these organisms. In this study, an existing in vivo spectrofluorometric assay method based on quantifying the cytochrome P450 mediated conversion of 7-ethocycoumarin (EtC) used as substrate to the product 7-hydroxycoumarin (HCm) called: ethoxycoumarin-O-deethylase (ECOD) activity, initially applicable on pooled samples of Daphnia magna, was optimized for use on individual organisms. Optimal assay conditions have been established for as small as 3- and 6 days old individuals, and the limits of spectrofluorometric detection of HCm excreted by daphnids in the incubation media were defined. The modified assay was tested by screening the modulation of ECOD activity in daphnids following 24 h exposure to ß-naphthoflavone (ß-NF, reference CYP450 inducer) and to prochloraz (PCZ), a potent CYP450 inhibitor. Maximal ECOD activity levels in daphnids were recorded following 2 hours of incubation to 200 nM EtC. The limit of spectrofluorometric detection of HCm in the incubation media was 6.25 nM, achieved by more than 80% of three days old daphnids and all six days old individuals. Exposure of daphnids to ß-NF demonstrated a bell-shaped ECOD activity induction potential, while PCZ elicited partial (60%) inhibition of ECOD activity. This optimized in vivo ECOD activity assay may serve as a cost-effective tool to study the responsiveness of Phase-I metabolism in D. magna to toxic pressure and its applicability to other aquatic invertebrates is also worth for consideration.

Presence, variation, and potential ecological impact of microplastics in the largest shallow lake of Central Europe.

The presence of microplastics (MPs) in the global ecosystem has generated a rapidly growing concern worldwide. Although their presence in the marine environment has been well-studied, much less data are available on their abundance in freshwaters. MPs alone and in combination with different chemicals has been shown to cause acute and chronic effects on algae and aquatic invertebrate and vertebrate species at different biological levels. However, the combined ecotoxicological effects of MPs with different chemicals on aquatic organisms are still understudied in many species and the reported data are often controversial. In the present study, we investigated, for the first time, the presence of MPs in Lake Balaton, which is the largest shallow lake of Central Europe and an important summer holiday destination. Moreover, we exposed neonates of the well-established ecotoxicological model organism Daphnia magna to different MPs (polystyrene [3 µm] or polyethylene [≤ 100 µm]) alone and in combination with three progestogen compounds (progesterone, drospirenone, levonorgestrel) at an environmentally relevant concentration (10 ng L-1) for 21 days. The presence of 7 polymer types of MPs in the size range of 50-100 µm was detected in Lake Balaton. Similarly to the global trends, polypropylene and polyethylene MPs were the most common types of polymer. The calculated polymer-independent average particle number was 5.5 particles m-3 (size range: 50 µm - 100 µm) which represents the values detected in other European lakes. Our ecotoxicological experiments confirmed that MPs and progestogens can affect D. magna at the behavioral (body size and reproduction) and biochemical (detoxification-related enzyme activity) levels. The joint effects were negligible. The presence of MPs may lead to reduced fitness in the aquatic biota in freshwaters such as Lake Balaton, however, the potential threat of MPs as vectors for progestogens may be limited.

Temperature related toxicity features of acute acetamiprid and thiacloprid exposure in Daphnia magna and implications on reproductive performance.

This study investigated the potential for elevated temperature to alter the toxicity of acetamiprid (ACE) and thiacloprid (Thia) in the ecotoxicity model Daphnia magna. The modulation of CYP450 monooxygenases (ECOD), ABC transporter activity (MXR) and incident cellular reactive oxygen species (ROS) overproduction was screened in premature daphnids following acute (48 h) exposure to sublethal concentrations of ACE and Thia (0.1-, 1.0 µM) at standard 21 °C and elevated 26 °C temperatures. Delayed outcomes of acute exposures were further evaluated based on the reproduction performance of daphnids monitored over 14 days of recovery. Exposures to ACE and Thia at 21o C elicited moderate induction of ECOD activity, pronounced inhibition of MXR activity and severe ROS overproduction in daphnids. In the high thermal regime, treatments resulted in significantly lower induction of ECOD activity and inhibition of MXR activity, suggesting a suppressed metabolism of neonicotinoids and less impaired membrane transport activity in daphnids. Elevated temperature on its own, caused a three-fold rise in ROS levels in control daphnids, while ROS overproduction upon neonicotinoid exposure was less accentuated. Acute exposures to ACE and Thia caused significant decreases also in the reproduction of daphnids, indicating delayed outcomes even at environmentally relevant concentrations. Both the cellular alterations in exposed daphnids and decreases in their reproductive output post exposures evidenced closely similar toxicity patterns and potentials for the two neonicotinoids. While elevated temperature elicited only a shift in baseline cellular alterations evoked by neonicotinoids, it significantly worsened the reproductive performance of daphnids following neonicotinoid exposures.

Chronic Effects of Carbamazepine, Progesterone and Their Mixtures at Environmentally Relevant Concentrations on Biochemical Markers of Zebrafish (Danio rerio).

The impact of pharmaceuticals on non-target organisms in the environment is of increasing concern and study. Pharmaceuticals and other pollutants are often present as mixtures in an environmental compartment. Studies on the toxicological implications of these drugs on fish, particularly as mixtures at environmentally relevant concentrations, are very limited. Thus, this study aimed to evaluate the chronic effects of the anticonvulsant drug carbamazepine (CBZ) and progesterone (P4) at environmentally relevant concentrations, individually and in binary mixtures, applying a suite of biomarkers at the molecular level in zebrafish (Danio rerio). The effects on biotransformation enzymes 7-ethoxyresorufin O-deethylase (EROD) and glutathione-S-transferase (GST), antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidases (GPxSe and GPxTOT), and glutathione reductase (GR), and markers of damage, such as DNA strand breaks (DNAsb), lactate dehydrogenase (LDH), lipid peroxidation (LPO), and vitellogenin-like proteins (VTG), were evaluated. Analyses of the biochemical markers indicated that a synergistic dose-ratio-dependent effect of CBZ and P4 in zebrafish occurs after chronic exposure regarding VTG, biotransformation enzymes (EROD, GST), and oxidative stress marker (DNAsb). The results suggest a synergistic effect regarding VTG, thus indicating a high risk to the reproductive success of fish if these pharmaceuticals co-occur.

Biochemical Marker Assessment of Chronic Carbamazepine Exposure at Environmentally Relevant Concentrations in Juvenile Common Carp (Cyprinus carpio).

Worldwide, the anticonvulsant drug carbamazepine (CBZ) is the most frequently identified pharmaceutical residue detected in rivers. Reported chronic effects of CBZ in non-target freshwater organisms, particularly fish, include oxidative stress and damage to liver tissues. Studies on CBZ effects in fish are mostly limited to zebrafish and rainbow trout studies. Furthermore, there are only a few chronic CBZ studies using near environmental concentrations. In this study, we provide data on subacute effects of CBZ exposure (28 days) to common carp (Cyprinus carpio), employing a set of biochemical markers of damage and exposure. CBZ was found to induce a significant change in the hepatic antioxidant status of fish subjected to 5 µg/L. Moreover, with increasing concentrations, enzymatic and non-enzymatic biomarkers of oxidative defence (catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), DNA strand breaks)), toxicant biotransformation (ethoxyresorufin-o-demethylase (EROD), glutathione-S-transferase (GST)), and organ and tissue damage (lactate dehydrogenase (LDH), cetylcholinesterase (AChE)) were altered. The AChE, LDH, and lipid peroxidation (LPO) results indicate the occurrence of apoptotic process activation and tissue damage after 28 days of exposure to CBZ. These findings suggest significant adverse effects of CBZ exposure to common carp at concentrations often found in surface waters.

The Role of IgG Fc Region N-Glycosylation in the Pathomechanism of Rheumatoid Arthritis.

Anti-citrullinated protein antibodies (ACPAs) are involved in the pathogenesis of rheumatoid arthritis. N-glycosylation pattern of ACPA-IgG and healthy IgG Fc differs. The aim of this study is to determine the relative sialylation and galactosylation level of ACPAs and control IgG to assess their capability of inducing TNFα production, and furthermore, to analyze the correlations between the composition of Fc glycans and inflammatory markers in RA. We isolated IgG from sera of healthy volunteers and RA patients, and purified ACPAs on a citrulline-peptide column. Immunocomplexes (IC) were formed by adding an F(ab)2 fragment of anti-human IgG. U937 cells were used to monitor the binding of IC to FcγR and to trigger TNFα release determined by ELISA. To analyze glycan profiles, control IgG and ACPA-IgG were digested with trypsin and the glycosylation patterns of glycopeptides were analyzed by determining site-specific N-glycosylation using nano-UHPLC-MS/MS. We found that both sialylation and galactosylation levels of ACPA-IgG negatively correlate with inflammation-related parameters such as CRP, ESR, and RF. Functional assays show that dimerized ACPA-IgG significantly enhances TNFα release in an FcγRI-dependent manner, whereas healthy IgG does not. TNFα production inversely correlates with the relative intensities of the G0 glycoform, which lacks galactose and terminal sialic acid moieties.

Comparison Evaluation of the Biological Effects of Sterigmatocystin and Aflatoxin B1 Utilizing SOS-Chromotest and a Novel Zebrafish (Danio rerio) Embryo Microinjection Method.

Aflatoxin B1 (AFB1) is a potent mycotoxin and natural carcinogen. The primary producers of AFB1 are Aspergillus flavus and A. parasiticus. Sterigmatocystin (STC), another mycotoxin, shares its biosynthetic pathway with aflatoxins. While there are abundant data on the biological effects of AFB1, STC is not well characterised. According to published data, AFB1 is more harmful to biological systems than STC. It has been suggested that STC is about one-tenth as potent a mutagen as AFB1 as measured by the Ames test. In this research, the biological effects of S9 rat liver homogenate-activated and non-activated STC and AFB1 were compared using two different biomonitoring systems, SOS-Chromotest and a recently developed microinjection zebrafish embryo method. When comparing the treatments, activated STC caused the highest mortality and number of DNA strand breaks across all injected volumes. Based on the E. coli SOS-Chromotest, the two toxins exerted the same genotoxicities. Moreover, according to the newly developed zebrafish microinjection method, STC appeared more toxic than AFB1. The scarce information correlating AFB1 and STC toxicity suggests that AFB1 is a more potent genotoxin than STC. Our findings contradict this assumption and illustrate the need for more complex biomonitoring systems for mycotoxin risk assessment.

Embryonic exposure to low concentrations of aflatoxin B1 triggers global transcriptomic changes, defective yolk lipid mobilization, abnormal gastrointestinal tract development and inflammation in zebrafish.

Aflatoxin B1-contaminated feeds and foods induce various health problems in domesticated animals and humans, including tumor development and hepatotoxicity. Aflatoxin B1 also has embryotoxic effects in different livestock species and humans. However, it is difficult to distinguish between the indirect, maternally-mediated toxic effects and the direct embryotoxicity of aflatoxin B1 in mammals. In the present study, we investigated the aflatoxin B1-induced direct embryotoxic effects in a zebrafish embryo model system combining toxicological, transcriptomic, immunological, and biochemical approaches. Embryonic exposure to aflatoxin B1 induced significant changes at the transcriptome level resulting in elevated expression of inflammatory gene network and repression of lipid metabolism and gastrointestinal tract development-related gene sets. According to the gene expression changes, massive neutrophil granulocyte influx, elevated nitric oxide production, and yolk lipid accumulation were observed in the abdominal region of aflatoxin B1-exposed larvae. In parallel, aflatoxin B1-induced defective gastrointestinal tract development and reduced L-arginine level were found in our model system. Our results revealed the complex direct embryotoxic effects of aflatoxin B1, including inhibited lipid utilization, defective intestinal development, and inflammation.

Toxicity mitigation by N-acetylcysteine and synergistic toxic effect of nano and bulk ZnO to Panagrellus redivivus.

To better understand the nanosize-relevant toxic effects and underlying mechanisms, N-acetylcysteine (NAC), as a mitigation agent, an ionic form of Zn (ZnCl2), and the binary mixture of ZnO with different particle sizes (15 nm and 140 nm), was used in toxicity assays with the nematode Panagrellus redivivus. The ZnCl2 concentrations were applied to show the amount of dissolved Zn ions present in the test system. Reactive oxygen species (ROS) measuring method was developed to fit the used test system. Our studies have shown that NAC can mitigate the toxic effects of both studied particle sizes. In the applied concentrations, ZnCl2 was less toxic than both of the ZnO particles. This finding indicates that not only ions and ROS produced by the dissolution are behind the toxic effects of the ZnO NPs, but also other particle size-dependent toxic effects, like the spontaneous ROS generation, are also relevant. When the two materials were applied in binary mixtures, the toxic effects increased significantly, and the dissolved zinc content and the ROS generation also increased. It is assumed that the chemical and physical properties of the materials have been mutually reinforcing to form a more reactive mixture that is more toxic to the P. redivivus test organism. Our findings demonstrate the importance of using mitigation agent and mixtures to evaluate the size-dependent toxicity of the ZnO.

Label-Free Semiquantitative Liquid Chromatography-Tandem Mass Spectrometry Proteomics Analysis of Laryngeal/Hypopharyngeal Squamous Cell Carcinoma on Formalin-Fixed, Paraffin-Embedded Tissue Samples - a Pilot Study.

Squamous cell carcinoma (SCC) of the head and neck region is the sixth most frequent malignancy with high mortality rate. Due to its poor prognosis it is considered a growing public health problem worldwide inspite of existing treatment modalities. Thus, early diagnosis of new diseases and recurrences is emerging on one hand, but on the other hand troublesome in the lack of reliable tumor markers in this field. The rapid development of proteomics has opened new perspectives in tumor marker discovery. Liquid chromatography/mass spectrometry (LC/MS) as the gold standard in proteomics enables the semi-quantitative analysis of proteins within various tissues. Abundance differences between tumor and normal tissue also can be interpreted as tumor specific changes. The aim of this study was to identify potential tumor markers of laryngeal/hypopharyngeal SCC by revealing abundance changes between cancerous and the surrounding phenotypically healthy tissue. After separating the phenotypically cancerous and healthy parts of formalin-fixed paraffin-embedded tissues, each sample underwent protein recovery process and tryptic digestion for label-free semi-quantitative LC/MS analysis. Eight proteins showed significantly higher abundance in tumor including tenascin, transmembrane emp24 domain-containing protein 2, cytoplasmic dynein light chain 1, coactosin-like protein, small proline-rich protein 2D, nucleolin, U5 small nuclear RNP 200-kDa helicase and fatty aldehyde dehydrogenase. Desmoglein-1 and keratin type I cytoskeletal 9 were down-regulated in tumor. Using Ingenuity Pathway Analysis we mapped the signaling pathways these proteins play role in regarding other tumors. Based on these findings these proteins may serve as promising biomarkers in the fight against laryngeal/hypopharyngeal SCCs.

Partial proteolysis improves the identification of the extracellular segments of transmembrane proteins by surface biotinylation.

Transmembrane proteins (TMP) play a crucial role in several physiological processes. Despite their importance and diversity, only a few TMP structures have been determined by high-resolution protein structure characterization methods so far. Due to the low number of determined TMP structures, the parallel development of various bioinformatics and experimental methods was necessary for their topological characterization. The combination of these methods is a powerful approach in the determination of TMP topology as in the Constrained Consensus TOPology prediction. To support the prediction, we previously developed a high-throughput topology characterization method based on primary amino group-labelling that is still limited in identifying all TMPs and their extracellular segments on the surface of a particular cell type. In order to generate more topology information, a new step, a partial proteolysis of the cell surface has been introduced to our method. This step results in new primary amino groups in the proteins that can be biotinylated with a membrane-impermeable agent while the cells still remain intact. Pre-digestion also promotes the emergence of modified peptides that are more suitable for MS/MS analysis. The modified sites can be utilized as extracellular constraints in topology predictions and may contribute to the refined topology of these proteins.

Covalently modified carboxyl side chains on cell surface leads to a novel method toward topology analysis of transmembrane proteins.

The research on transmembrane proteins (TMPs) is quite widespread due to their biological importance. Unfortunately, only a little amount of structural data is available of TMPs. Since technical difficulties arise during their high-resolution structure determination, bioinformatics and other experimental approaches are widely used to characterize their low-resolution structure, namely topology. Experimental and computational methods alone are still limited to determine TMP topology, but their combination becomes significant for the production of reliable structural data. By applying amino acid specific membrane-impermeable labelling agents, it is possible to identify the accessible surface of TMPs. Depending on the residue-specific modifications, new extracellular topology data is gathered, allowing the identification of more extracellular segments for TMPs. A new method has been developed for the experimental analysis of TMPs: covalent modification of the carboxyl groups on the accessible cell surface, followed by the isolation and digestion of these proteins. The labelled peptide fragments and their exact modification sites are identified by nanoLC-MS/MS. The determined peptides are mapped to the primary sequences of TMPs and the labelled sites are utilised as extracellular constraints in topology predictions that contribute to the refined low-resolution structure data of these proteins.

Site-specific N-glycosylation of HeLa cell glycoproteins.

We have characterized site-specific N-glycosylation of the HeLa cell line glycoproteins, using a complex workflow based on high and low energy tandem mass spectrometry of glycopeptides. The objective was to obtain highly reliable data on common glycoforms, so rigorous data evaluation was performed. The analysis revealed the presence of a high amount of bovine serum contaminants originating from the cell culture media - nearly 50% of all glycans were of bovine origin. Unaccounted, the presence of bovine serum components causes major bias in the human cellular glycosylation pattern; as is shown when literature results using released glycan analysis are compared. We have reliably identified 43 (human) glycoproteins, 69 N-glycosylation sites, and 178 glycoforms. HeLa glycoproteins were found to be highly (68.7%) fucosylated. A medium degree of sialylation was observed, on average 46.8% of possible sialylation sites were occupied. High-mannose sugars were expressed in large amounts, as expected in the case of a cancer cell line. Glycosylation in HeLa cells is highly variable. It is markedly different not only on various proteins but also at the different glycosylation sites of the same protein. Our method enabled the detailed characterization of site-specific N-glycosylation of several glycoproteins expressed in HeLa cell line.

Identification of bifucosylated glycoforms using low-energy CID spectra.

We have used tandem mass spectrometry (MS/MS)-based analysis of glycopeptides in order to identify the composition and structure of rare glycoforms. The results illustrate utility of low-energy MS/MS for structure identification. We have shown the presence of bifucosylated and trifucosylated glycoforms in human α-1-acid glycoprotein (AGP), a major plasma glycoprotein. Fucosylation in the case of AGP always occurs on the antennae; core fucosylation was not observed.

Unravelling the Role of Trophoblastic-Derived Extracellular Vesicles in Regulatory T Cell Differentiation.

Regulatory T cells (Treg) are mandatory elements in the maintenance of human pregnancy, but their de novo differentiation has not been completely exposed. HSPE1 chaperone expressing trophoblast cells may have a role in it. Trophoblast-derived extracellular vesicles (EVs), either at the feto-maternal interface or in circulation, target CD4+ T cells. We hypothesized that HSPE1-associated trophoblastic cell line (BeWo)-derived EVs are active mediators of Treg cell differentiation. We proved at first that recombinant HSPE1 promote human Treg cell differentiation in vitro. Developing a CRISPR-Cas9 based HSPE1 knockout BeWo cell line we could also demonstrate, that EV-associated HSPE1 induces Treg development. Next-generation sequencing of miRNA cargo of BeWo-EVs characterized the regulatory processes of Treg polarization. By the use of single-cell transcriptomics analysis, seven Treg cell subtypes were distinguished and we demonstrated for the first time that the expression level of HSPE1 was Treg subtype dependent, and CAPG expression is characteristic to memory phenotype of T cells. Our data indicate that HSPE1 and CAPG may be used as markers for identification of Treg subtypes. Our results suggest, that trophoblastic-derived iEVs-associated HSPE1 and miRNA cargo have an important role in Treg cell expansion in vitro and HSPE1 is a useful marker of Treg subtype characterization.

High sensitivity proteomics of prostate cancer tissue microarrays to discriminate between healthy and cancerous tissue.

Biopsies, in the form of tissue microarrays (TMAs) were studied to identify anomalies indicative of prostate cancer at the proteome level. TMAs offer a valuable source of well-characterized biological material. However, because of the small tissue sample size method development was essential to provide the sensitivity and reliability necessary for the analysis. Surface digestion of TMA cores was followed by peptide extraction and shotgun proteomics analysis. About 5 times better sensitivity was achieved by the optimized surface digestion compared to bulk digestion of the same TMA spot and it allowed the identification of over 500 proteins from individual prostate TMA cores. Label-free quantitation showed that biological variability among all samples was about 3 times larger than the technical reproducibility. We have identified 189 proteins which showed statistically significant changes (t-test p-value <.05) in abundance between healthy and cancerous tissue samples. The proteomic profile changed according to cancer grade, but did not show a correlation with cancer stage. Results of this pilot study were further evaluated using bioinformatics tools, identifying various protein pathways affected by prostate cancer progression indicating the usefulness of studying TMA cores to identify quantitative changes in tissue proteomics. SIGNIFICANCE: Detailed proteomics analysis of TMAs presents a good alternative for tissue analysis. Here we present a novel method, based on tissue surface digestion and nano-LC-MS measurements, which is capable of identifying and quantifying over 500 proteins from a 1.5 mm diameter tissue section. We compared healthy and cancerous prostate tissue samples, and tissues with various grades and stages of cancer. Tissue proteomics clearly distinguished healthy and cancerous samples, furthermore the results correlated well with cancer grade, but not with cancer stage. Over 100 proteins showed statistically significant abundance changes (t-test p-value <.05) between various groups. This was sufficient for a meaningful bioinformatics evaluation; showing e.g. increased abundance of proteins in cancer in the KEGG ribosome pathway, GO mRNA splicing via spliceosome, and chromatin assembly biological processes. The results highlight the feasibility of the developed method for future large-scale tissue proteomics studies using commercially available TMAs.

Distinguishing Core and Antenna Fucosylated Glycopeptides Based on Low-Energy Tandem Mass Spectra.

A straightforward approach has been developed to distinguish core and antenna fucosylation in glycopeptides. The method does not require derivatization and can be easily adapted into a proteomics workflow. The key aspect is to use low collision energy collision-induced dissociation (CID) (on a quadrupole time-of-flight type instrument) when only single-step fragmentation processes occur. Low collision energy should show the precursor ion as the largest peak in the spectrum; the survival yield should be ideally over 50%, and this is obtained at a collision energy ca. 30% of that typically used for proteomics. In such a case, interfering processes like fucose migration or consecutive reactions are minimized. Core and antenna fucosylation can be discriminated using various ion abundance ratios. Low-energy CID spectra are very "clean" (no chemical noise), and the ions used for locating the fucose are among the major peaks, making the method well-suited for analytical work. Monitoring the change in the proportion of core and antenna fucosylation at the same glycosylation site is also feasible.

Author Correction: The impact of circulating preeclampsia-associated extracellular vesicles on the migratory activity and phenotype of THP-1 monocytic cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

The impact of circulating preeclampsia-associated extracellular vesicles on the migratory activity and phenotype of THP-1 monocytic cells.

Intercellular communication via extracellular vesicles (EVs) and their target cells, especially immune cells, results in functional and phenotype changes that consequently may play a significant role in various physiological states and the pathogenesis of immune-mediated disorders. Monocytes are the most prominent environment-sensing immune cells in circulation, skilled to shape their microenvironments via cytokine secretion and further differentiation. Both the circulating monocyte subset distribution and the blood plasma EV pattern are characteristic for preeclampsia, a pregnancy induced immune-mediated hypertensive disorder. We hypothesized that preeclampsia-associated EVs (PE-EVs) induced functional and phenotypic alterations of monocytes. First, we proved EV binding and uptake by THP-1 cells. Cellular origin and protein cargo of circulating PE-EVs were characterized by flow cytometry and mass spectrometry. An altered phagocytosis-associated molecular pattern was found on 12.5 K fraction of PE-EVs: an elevated CD47 "don't eat me" signal (p < 0.01) and decreased exofacial phosphatidylserine "eat-me" signal (p < 0.001) were found along with decreased uptake of these PE-EVs (p < 0.05). The 12.5 K fraction of PE-EVs induced significantly lower chemotaxis (p < 0.01) and cell motility but accelerated cell adhesion of THP-1 cells (p < 0.05). The 12.5 K fraction of PE-EVs induced altered monocyte functions suggest that circulating EVs may have a role in the pathogenesis of preeclampsia.

Sensitive method for glycosaminoglycan analysis of tissue sections.

A simple, isocratic HPLC method based on HILIC-WAX separation, has been developed for analyzing sulfated disaccharides of glycosaminoglycans (GAGs). To our best knowledge, this is the first successful attempt using this special phase in nano-HPLC-MS analysis. Mass spectrometry was based on negative ionization, improving both sensitivity and specificity. Detection limit for most sulfated disaccharides were approximately 1 fmol; quantitation limits 10 fmol. The method was applied for glycosaminoglycan profiling of tissue samples, using surface digestion protocols. This novel combination provides sufficient sensitivity for GAG disaccharide analysis, which was first performed using prostate cancer tissue microarrays. Preliminary results show that GAG analysis may be useful for identifying cancer related changes in small amounts of tissue samples (ca. 10 µg).