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Exosomes have been implicated in numerous biological processes, and they may serve as important disease markers. Surface proteins on exosomes carry information about their tissues of origin. Because of the heterogeneity of exosomes it is desirable to investigate them individually, but this has so far remained impractical. Here, we demonstrate a proximity-dependent barcoding assay to profile surface proteins of individual exosomes using antibody-DNA conjugates and next-generation sequencing. We first validate the method using artificial streptavidin-oligonucleotide complexes, followed by analysis of the variable composition of surface proteins on individual exosomes, derived from human body fluids or cell culture media. Exosomes from different sources are characterized by the presence of specific combinations of surface proteins and their abundance, allowing exosomes to be separately quantified in mixed samples to serve as markers for tissue-specific engagement in disease.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture "what the community needs in a tool". Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

Objective: To examine the correlation between plasma hCG results obtained with the new i-STAT® hCG point of care test with those concomitantly obtained from the central hospital laboratory utilizing the same patient samples. Methods: Prospective cross-sectional laboratory test evaluation. We compared plasma hCG results obtained with the i-STAT® hCG test (Abbott Point of Care, Princeton, NJ, USA) with Architect Ci8200 (Abbott Laboratories, Abbott Park, IL, USA). We also calculated the total coefficient of variation (CV) for the i-STAT® method. Results: The two methods showed a good linear correlation (R2 = 0.994; slope 1.03) and CV for the i-STAT® method was 2.1% - 5.2%. Conclusion: We suggest that the i-STAT® hCG blood assay could be used as a complement to urine hCG assays in clinical situations when rapid test results are needed and urine is not available.

Cancer is a leading cause of thrombosis. We identify a new procoagulant mechanism that contributes to thromboembolism in prostate cancer and allows for safe anticoagulation therapy development. Prostate cancer-mediated procoagulant activity was reduced in plasma in the absence of factor XII or its substrate of the intrinsic coagulation pathway factor XI. Prostate cancer cells and secreted prostasomes expose long chain polyphosphate on their surface that colocalized with active factor XII and initiated coagulation in a factor XII-dependent manner. Polyphosphate content correlated with the procoagulant activity of prostasomes. Inherited deficiency in factor XI or XII or high-molecular-weight kininogen, but not plasma kallikrein, protected mice from prostasome-induced lethal pulmonary embolism. Targeting polyphosphate or factor XII conferred resistance to prostate cancer-driven thrombosis in mice, without increasing bleeding. Inhibition of factor XII with recombinant 3F7 antibody reduced the increased prostasome-mediated procoagulant activity in patient plasma. The data illustrate a critical role for polyphosphate/factor XII-triggered coagulation in prostate cancer-associated thrombosis with implications for anticoagulation without therapy-associated bleeding in malignancies.

Background: Endostatin is an endogenous inhibitor of angiogenesis that inhibits neovascularisation. The aim of the study was to evaluate the effect of elective surgery on endostatin levels. Methods: Blood samples were collected prior to elective surgery and 4 and 30 days postoperatively in 2 patient groups: orthopedic surgery (n =27) and coronary bypass patients (n =21). Serum endostatin levels were measured by ELISA. Results: Serum endostatin was significantly reduced 30 days after surgery in comparison with presurgical values in both the orthopedic (P =0.03) and cardiopulmonary surgery (P =0.04) group. Conclusion: Serum endostatin is reduced 30 days after surgery. This reduction would favor angiogenesis and wound-healing.

Release of nanometer-sized prostasomes into human and equine semen suggests essential functions in their relationships with sperm cells and the fertilization process. The two types of prostasomes displayed ultrastructural similarities, albeit the human prostasomes were somewhat larger than the stallion prostasomes. A high ratio of saturated fatty acids was characteristic for the two prostasome types. Electrophoretic separation systems revealed an equine prostasomal pattern different from that of human. The 21 distinctive low molecular weight protein spots in the 2D-gel (with no counterparts in human prostasomes) were identified via peptide mass fingerprinting, several of which may be different isoforms. Out of the three high molecular weight bands characteristic for human prostasomes (CD10, CD13, and CD26), CD10 and CD13 were retrieved in equine prostasomes. We present some new proteins of horse prostasomes not found in their human counterparts. Further studies are warranted to reveal the function of these proteins.

Objectives. Pentraxin 3 (PTX3) is an acute phase marker, which is produced at the site of infection or inflammation in contrast to CRP that is mainly synthesized by the liver. The aim of the present study was to see if lifestyle interventions/weight loss would lead to decreased blood plasma concentrations of PTX3. Methods. Study subjects (n = 31) were recruited to a lifestyle intervention program aiming at increased physical activity, improved eating habits, and weight loss. High-sensitivity C-reactive protein (CRP) and PTX3 methods were used for analysis of CRP and PTX3 in plasma samples collected at inclusion and after 4 and 8 weeks of treatment. Results. Wilcoxon paired samples test showed a significant decrease in PTX3 concentrations from 2068 pg/mL at start to 2007 pg/mL at 4 weeks (P = 0.002) and 1748 pg/mL at 8 weeks (P = 0.003). The PTX3 decrease was not significantly correlated with a corresponding decrease in CRP or weight reduction. Conclusions. The lifestyle intervention program resulted in a significant reduction of circulating concentrations of pentraxin 3 already after 4 and 8 weeks of treatment.

Semen is a heterogenous and complex fluid with different functions, some of them well known, others still obscure. The aim of this study was to investigate the presence of cathepsins B, S and L in human seminal plasma and their possible associations with other semen variables. Cathepsin B, L and S concentrations were measured in seminal plasma from 99 men utilising commercial ELISA kits. Seminal plasma cathepsin B was approximately 70 times higher, while the cathepsin L values were approximately 500 times higher and the cathepsin S values approximately 40 times higher in seminal plasma than in a group of serum samples. The study shows that seminal plasma contains high levels of cathepsins B, L and S. All three cathepsins were also bound to the surface of prostasomes.

BACKGROUND: Prostate acinar epithelial cells release microvesicles (prostasomes) that possess pleiotropic biological effects relevant for successful fertilization. Prostasomes are formed in a similar way as exosomes but are heterogeneous as regards size and appearance. Like exosomes they are thought to be mediators of intercellular communication. METHODS: We prepared seminal prostasomes in accordance with the prevailing protocol for exosome preparation including passage through a 0.2 µm filter and centrifugation in a sucrose gradient. RESULTS: We compared the "filterable prostasomes" with those trapped on the filter ("nonfilterable prostasomes") and, qualitatively, no conspicuous differences were apparent regarding ultrastructure and SDS-PAGE banding pattern. Moreover, both types of prostasomes contained DNA fragments and Western blot revealed presence of prostate specific membrane antigen (PSMA), CD38, and annexin A1. CONCLUSIONS: Reasonably, prostasomes could be included in the exosome family and be regarded as one entity containing chromosomal DNA.

Human prostasomes, exosome-like microvesicles secreted by acinar cells of the prostate gland, contain chromosomal DNA. Agarose gel electrophoresis of DNA from seminal prostasomes displayed fragments of over 12 kb and smaller, with a distinct band around 1 kb that was excised, cloned, and sequenced. The sequences showed 8 out of 25 clones (32%) originating from genes. We elaborated the concept further by carrying out a genome-wide DNA copy number analysis of prostasomal DNA, hypothesizing that human prostasomes contain fragments of DNA randomly selected from the entire genome. Acridine orange-stained prostasomes were incubated with freshly prepared sperm for different times, and a transfer of acridine orange-stained prostasomal DNA to sperm (preferentially the head region) was observed. Fluorescence microscopy of slices in the center of 14 optical slides of the sperm head displayed an even fluorescence rather than a halo-like one, indicating DNA-uptake rather than just binding along the sperm head membrane.

The secretory epithelial cells of the prostate gland use sophisticated vehicles in the form of prostasomes to relay important information to sperm cells in semen. This prostasome-forming and secretory ability of the epithelial cells is also preserved in poorly differentiated prostate cancer cells. We investigated prostasomes from vertebral metastases of prostate cancer, taken from the operating field at surgery, directly taken care of under protease inhibitory conditions for later 2-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) protein characterization. A total of 104 spots were punched out for identification. Twenty five unique protein spots had a MALDI-TOF above 49 and another 5 proteins were determined by MS/MS. The remaining 74 spots were either identical to already determined proteins or had no reliable score. Annexins A1, A3, and A5 as well as dimethylarginine dimethylaminohydrolase 1 were among the identified proteins. The annexins and dimethylarginine dimethylaminohydrolase 1 found in cancer-derived prostasomes can act, among other things, as angiogenic factors and can increase the vascular development in the neighborhood of the tumor. Cancer-derived prostasomes may play an important role in the interaction between tumor cells and their environment.

BACKGROUND. The aim of this study was to perform a comprehensive evaluation of the occurrence of DNA in human prostasomes. METHODS. Prostasomes were purified from seminal fluid (seminal prostasomes) and from PC-3-cells (PC-3 cell prostasomes). DNA extracted from both sources of prostasomes was visualized on agarose gels. Further, theDNAwas cloned and sequenced (13 clones from seminal prostasomal DNAand 16 clones from PC-3 cell prostasomal DNA) and identified by alignment in the BLAST-nucleotide search database. In order to decide if the DNA was internally or externally located in/on prostasomes, prostasomes were treated with nuclease (DNase) and A260 was measured before and after treatment. Additionally, flow cytometric studies were performed with membrane permeable and membrane impermeable DNA stains. RESULTS. We identified human chromosomal DNA in purified prostasomes from both sources and treatment with DNase demonstrated that the prostasome-shielded DNA was protected from enzyme attack. Membrane-permeable DNA stain raised the fluorescence contrary to membrane-impermeable stain. Clearly discernible nucleic acid of prostasomes was separated on 1% agarose gel yieldingDNAfragments of about 13 kbp and below with a marked band at about 1 kbp. Cloning and sequencing of 13 fragments from seminal prostasomes and 16 from PC-3 cell prostasomes revealed a chromosomal origin of the DNA. In purified seminal prostasomes, 4 out of 13 DNA clones featured gene sequences (31%). The corresponding figure for PC3-derived prostasomes was 4 out of 16 clones featuring gene sequences (25%). CONCLUSION. Human prostasomes contain chromosomal DNA. Both nuclease treatment and differential DNA stainings indicated an inside location of the prostasomal DNA. Our findings suggest a DNA-delivery function of prostasomes to sperm cells.

Objective. Clusterin is a ubiquitous secretory sulphated glycoprotein present in prostasomes. It is an antiapoptotic mediator in prostate cancer and is among the most frequently occurring prostasomal proteins immunogenic in prostate cancer patients. The aim of the present study was to investigate the occurrence of anticlusterin antibodies in the serum of patients with prostate cancer and whether there is a relationship between anticlusterin antibody titres and other clinico-pathological variables. Material and methods. Serum samples were collected from 391 consecutive patients with suspected prostate cancer (150 benign prostate and 241 prostate cancer). The patients’ serum samples were used in an ELISA where microtitre wells were coated with purified clusterin from serum of a healthy volunteer. Flow cytometric studies of clusterin and prostasomes were performed. Results. Flow cytometric analyses revealed the presence of clusterin on the surface of seminal prostasomes. Anti-clusterin ELISA titres in sera of patients did not differ significantly from those of a control group. A significant ‘‘inverse’’ correlation existed between anti-clusterin ELISA titres and lymph node metastases (p50.047), but only 11 out of 161 patients had metastases. These titres correlated significantly with total prostate (p50.021) and transitional zone (p50.015) volumes of the patients. Conclusions. The correlation between serum anti-clusterin antibody titres and other clinico-pathological variables was generally weak in prostate cancer patients, although clusterin has been assigned an important role in tumourigenesis and progression of prostate cancer. However, the anti-clusterin antibody titre appeared to be related to prostate volume, correlating to both transitional zone volume and total volume of the prostate.

Prostate cancer consistently remains a difficult clinical enigma. Therefore, the development of novel strategies for diagnosis and treatment (e.g. immunotherapy) of prostate cancer is essential. We tried to identify the prostasome-derived proteins that were immunogenic in prostate cancer patients. Prostate cancer patients’ sera (n 5 44) with high enzyme-linked immunosorbent assay (ELISA) titers against prostasomes were selected for immunoblotting against purified seminal prostasomes. The SDS-PAGE and immunoblotting experiments were performed with Bio-Rad systems. Twenty-five of the recognized proteins were isolated and analyzed by means of mass spectrometry. Out of 44 patients’ sera, 31 (70%) demonstrated in immunoblotting experiments reactivity against several prostasomal protein bands in the molecular weight range of 10– 200 kDa. Some of the bands (55, 70 and 170 kDa) were more frequently recognized by the patients’ sera. Concomitantly run control sera generated only very weak or no bands at all. The most frequently occurring prostasomal proteins were identified as heat shock proteins (HSP 70, 71) and clusterin. This study identified the most important molecular targets of autoantibodies against prostasomes generated in connection with the development of prostate cancer in man. These immunogenic prostasomal proteins could be appropriate target molecules for specific immunotherapy of prostate cancer patients.

OBJECTIVE: To investigate the ability of human uterine myocytes to grow under anaerobic conditions for a prolonged time period. METHODS: Cells were isolated from fundal myometrium and cultured until subconfluency. The cell type was confirmed by immunostaining for the smooth muscle cell-specific cytoskeletal proteins alpha-actin and desmin. Some cells were further cultured under aerobic conditions and others under anaerobic conditions. Cells were harvested after 0, 4 and 8 days in culture and analyzed for their content of adenylates. RESULTS: Immunostaining revealed that all three preparations contained almost only smooth muscle cells. Energy charge of the myocytes was 0.88 on average at the beginning of the culture experiment. A moderate decrease was noted on day 4 for myocytes grown under both aerobic and anaerobic conditions and no further decrease was noted between days 4 and 8. Morphologically the cells retained their normal appearance and they seemed healthy for at least 8 days in culture under both aerobic and anaerobic conditions. CONCLUSIONS: The results of this study suggest that human myometrial cells can survive for an extended period of time under in vitro conditions regardless of oxygen availability for energy metabolism. This means that anaerobic energy metabolism is enough to sustain vital processes during that period of time.

The aim of the present study was to investigate possible relationships in piglets between myocardial energy-related metabolites and intracellular electrolytes during open-chest cardiopulmonary resuscitation (OCCPR) supplemented by the administration of alkaline buffers with varying sodium content. Our hypothesis was that an increasing myocardial intracellular sodium content would decrease the intracellular energy stores. In addition to haemodynamics, acid-base and blood gas variables were analysed, and myocardial biopsies were collected before and during OCCPR as well as after the return of spontaneous circulation. After a period of 4 min of untreated ventricular fibrillation (VF). 25 piglets were randomly allocated to one of four groups: OCCPR with normal saline (n = 5); OCCPR with sodium bicarbonate (SB) (n = 7); OCCPR with Tris buffer mixture (TBM) (n = 7); and a totally untreated control group (n = 6). The results showed that 4 min of untreated VF almost eradicated creatine phosphate (CrP) and that the ATP/ADP ratio decreased to 1.5-2.0. During OCCPR with normal saline, the myocardial content of CrP increased, whereas lactate, ATP and ADP levelled off and AMP decreased, causing an increased ATP/ADP ratio. The adenosine and inosine contents increased, whereas inosine monophosphate was unchanged at a low level, the adenosine and inosine contents being inversely correlated with the total content of adenine nucleotides. In both buffered groups, the increase in most energy-related metabolites (CrP, ATP, ADP, AMP and the ATP/ADP quotient) was less and in lactate more pronounced than in the group not being buffered, with no difference between the groups receiving SB or TBM. Although the intracellular potassium content was unaltered, the sodium, chloride and calcium concentration increased, more so in the group receiving SB. The intracellular content of sodium was correlated with that of calcium. Thus, buffering increased the myocardial AMP degradation during OCCPR by i