Proteomics Analysis of Bladder Cancer Exosomes

Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function.Bladder cancer is one of the eight most frequent cancers in the Western world, and the frequency of transitional cell carcinoma (TCC),¹ which accounts for 90% of bladder cancers, is second only to prostate cancer as a malignancy of the genitourinary tract. Urine cytology and cystoscopy remain the predominant clinical tools for diagnosing and monitoring the disease, but cytology is poorly sensitive, particularly for low grade tumors, and does not serve as a prognostic tool. Cystoscopy is an invasive procedure, and there is pressing need to identify informative molecular markers that can be used to replace it.Recently, small cell-derived vesicles termed exosomes that are present in body fluids (1–5) have been proposed as a potential source of diagnostic markers (2, 6–8). These nanometer-sized vesicles, which are secreted by most cell types, originate from multivesicular bodies of the endocytic tract and reflect a subproteome of the cell. Exosomes are enriched in membrane and cytosolic proteins, and this molecular repertoire appears to be of particular functional importance to the immune system (9). Exosomes also comprise an array of lipids, mRNA, and microRNA, which are likely involved in conveying intercellular communication processes (10). Importantly, many exosomal components are simply not present as free soluble molecules in body fluids, such as certain microRNA species, which are encapsulated within the exosome lumen (6, 10). Therefore, the ability to isolate exosomes from urine (2), plasma (1), saliva (11), or other physiological sources (3) holds significant potential for obtaining novel and complex sets of biomarkers in a non-invasive manner. Exosome analysis may therefore be of value in disease diagnosis and monitoring in a variety of settings (6, 7, 12–14).Exosomes as indicators of pathology were first documented in the context of renal injury where a differential proteomics approach revealed changes in urinary exosome phenotype following renal injury (7). The researchers identified exosomally expressed Fetuin-A as a marker that became elevated 50-fold within hours following nephrotoxin exposure in rodents. Exosomal Fetuin-A elevation was also apparent in patients with acute renal injury before changes in urinary creatinine were observed (7). Clinical exosome analysis may also prove useful for solid cancers, such as ovarian or lung cancer, where the quantity of epithelial cell adhesion molecule-positive serum exosomes may correlate with tumor stage/grade. Such disease-associated exosomes express microRNA species not detected in healthy subjects (6, 12), although in this respect, there is little correlation between microRNA and disease bulk (6, 12). Other recent examples include studies of urinary exosomes in prostate cancer with exosomes expressing protein markers 5T4 (15), prostate cancer gene 3 (PCA-3) (8), or mRNA (TMPRSS2-ERG) (8, 16) associated with prostate cancer. To our knowledge, exosomes have not yet been studied in the context of other urological malignancies such as renal cancer, and to date, only one report describes the urine-derived microparticles from bladder cancer patients (17). In that report, they examined the proteome of a highly complex mixture of microvesicles, exosomes, and other urinary constituents that can be pelleted by high speed ultracentrifugation, identifying eight proteins that may be elevated in cancer. However, given the nature of the sample analyzed, it is unknown whether these proteins are exosomally expressed.Identification of the principal and most relevant molecular markers in these and other clinical scenarios remains a major challenge. In part, this is because exosomes present within complex body fluids originate from heterogeneous cell types. For example, plasma exosomes may be derived from platelets, lymphocytes, or endothelial cells (1), and a proportion may arise from well perfused organs such as the liver (18) and likely other organs as well (16). Similarly, exosomes present in urine arise from urothelial cells of the kidney and downstream of the renal tract (2, 8, 15).Importantly, all proteomics studies of exosomes isolated from body fluids are unavoidably complicated by the presence of high abundance non-exosomal proteins contaminating the preparations. Examples include albumin, immunoglobulin, and complement components present in exosomes prepared from malignant effusions (5) and Tamm-Horsfall protein present in exosomes purified from urine (2). As such, great care must be taken in the interpretation of the large data sets produced by proteomics studies, requiring careful validation of the proteins of interest. The protein composition of exosomes using a single homogenous cell type is one approach that may be used to uncover the protein components of exosomes produced by various cell types.There remain two major issues in the realm of exosome proteomics that complicate our interpretation of lists of identified proteins. Foremost are the diverse methods chosen for exosome purification that in some studies have involved attempts to remove contaminants through a key biophysical property of the vesicles, i.e. their capacity to float on sucrose (19, 20) or other dense media (21). Not all published studies, however, have taken such steps, preferring a far simpler pellet (or pellet and wash) approach. These latter preparations may be significantly contaminated by components of the cellular secretome, cell fragments, and other components. All of these factors could lead to false positive identifications of exosome proteins. The second key issue centers on the MS approaches utilized in various exosome proteomics studies. Many early examples relied only on a peptide mass fingerprinting approach, lacking robust peptide sequence data (22, 23), and more recently, search criteria that are generally recommended for MS-derived sequence data have not been specified in all studies. In this study, we have listed only those proteins identified by good quality MS/MS data for two or more peptides. Variability in the robustness and bias in bioinformatics analysis of data sets and in the steps taken to validate identified proteins is an additional factor that impacts the confidence in the identification lists produced.In this study, we aimed to perform the first proteomics analysis of human bladder cancer exosomes. We took extensive steps to produce high purity and quality-assured exosome preparations prior to beginning proteomics workflows. Solubilizing the sample with SDS and a reducing agent (DTT) was a critical step that allowed for global protein identification using nanoscale liquid chromatography followed by MALDI-TOF/TOF mass spectrometry. In this study, we present the identification of a significant number of exosomally expressed proteins (353 in total) of unrivaled quality. Critical manual examination of these identifications revealed issues with multiple (physiologically impossible) MHC Class I identifications that were attributed to a misdesignation of nomenclature by MASCOT due to peptide (and target protein) homology. The data were subjected to unbiased overrepresentation analysis (examining ExoCarta and Gene Ontology databases) to reveal a proteome consistent with exosomes, particularly of carcinoma origin. Validation of several identified proteins, by combining ultracentrifugation on a linear sucrose gradient with Western blotting and/or analysis of exosome-coated latex beads, demonstrated correct surface orientation of several MS-identified membrane proteins at densities consistent with exosomes.The robust approaches taken emphasize our confidence in the validity of the identifications generated and highlight that 72 (of 353) proteins have not been previously shown to be exosomally expressed by other human proteomics studies. The data will be useful for future studies in this underinvestigated disease and will form a platform not only for future clinical validation of some of these putative markers but also to aid further investigations into novel aspects of exosome function and manufacture.     

A conodont, thelodont and acanthodian fauna from the Lower Přídolí (Silurian) of the Much Wenlock area, Shropshire

Conodonts, thelodonts and acanthodians have previously been collected from the P?ídolí of Shropshire (Welsh Borderland) from bone beds such as the Ludlow Bone Bed. In general, specimens collected from these types of deposit are very abraded and make taxonomic studies difficult. This contribution describes well preserved thelodont microelements from two samples from the Downton Castle Sandstone Formation (P?ídolí, Silurian) of the Much Wenlock area of Shropshire. Head scales, a wide variety of transitional scales, and pore scales of the lateral line sensory canals of the thelodont Paralogania ludlowiensis (Gross) are described for the first time. A new acanthodian Nostolepis linleyensis sp. nov. and elements from the apparatus of the conodont Ozarkodina?hemensis sp. nov. are documented from the same samples. The thelodonts in both samples are typical of upper Ludlow–P?ídolí faunas from across the Welsh Borderland, and are also present in smaller numbers in the Ludlow Series of Gotland, Sweden, Saaremaa Island, Estonia, and in Latvia and Lithuania. The conodont O? hemensis sp. nov. is restricted to the Much Wenlock area of Shropshire. The co-occurrence of well preserved elements of conodonts and thelodonts suggests that they were deposited fairly rapidly with little or no re-working and that a restricted marine environment prevailed in the early P?ídolí in the Much Wenlock area of Shropshire.     

Bayesian inference for stochastic epidemic models with time-inhomogeneous removal rates

Stochastic compartmental models of the SEIR type are often used to make inferences on epidemic processes from partially observed data in which only removal times are available. For many epidemics, the assumption of constant removal rates is not plausible. We develop methods for models in which these rates are a time-dependent step function. A reversible jump MCMC algorithm is described that permits Bayesian inferences to be made on model parameters, particularly those associated with the step function. The method is applied to two datasets on outbreaks of smallpox and a respiratory disease. The analyses highlight the importance of allowing for time dependence by contrasting the predictive distributions for the removal times and comparing them with the observed data.      

Domestication and crop physiology: roots of green-revolution wheat

BACKGROUND AND AIMS: Most plant scientists, in contrast to animal scientists, study only half the organism, namely above-ground stems, leaves, flowers and fruits, and neglect below-ground roots. Yet all acknowledge roots are important for anchorage, water and nutrient uptake, and presumably components of yield. This paper investigates the relationship between domestication, and the root systems of landraces, and the parents of early, mid- and late green-revolution bread wheat cultivars. It compares the root system of bread wheat and 'Veery'-type wheat containing the 1RS translocation from rye. METHODS: Wheat germplasm was grown in large pots in sand culture in replicated experiments. This allowed roots to be washed free to study root characters. KEY RESULTS: The three bread wheat parents of early green-revolution wheats have root biomass less than two-thirds the mean of some landrace wheats. Crossing early green-revolution wheat to an F(2) of 'Norin 10' and 'Brevor', further reduced root biomass in mid-generation semi-dwarf and dwarf wheats. Later-generation semi-dwarf wheats show genetic variation for root biomass, but some exhibit further reduction in root size. This is so for some California and UK wheats. The wheat-rye translocation in 'Kavkaz' for the short arm of chromosome 1 (1RS) increased root biomass and branching in cultivars that contained it. CONCLUSIONS: Root size of modern cultivars is small compared with that of landraces. Their root system may be too small for optimum uptake of water and nutrients and maximum grain yield. Optimum root size for grain yield has not been investigated in wheat or most crop plants. Use of 1RS and similar alien translocations may increase root biomass and grain yield significantly in irrigated and rain-fed conditions. Root characters may be integrated into components of yield analysis in wheat. Plant breeders may need to select directly for root characters.     

Characterization of an acid-dependent arginine decarboxylase enzyme from Chlamydophila pneumoniae

Genome sequences from members of the Chlamydiales encode diverged homologs of a pyruvoyl-dependent arginine decarboxylase enzyme that nonpathogenic euryarchaea use in polyamine biosynthesis. The Chlamydiales lack subsequent genes required for polyamine biosynthesis and probably obtain polyamines from their host cells. To identify the function of this protein, the CPn1032 homolog from the respiratory pathogen Chlamydophila pneumoniae was heterologously expressed and purified. This protein self-cleaved to form a reactive pyruvoyl group, and the subunits assembled into a thermostable (alphabeta)(3) complex. The mature enzyme specifically catalyzed the decarboxylation of L-arginine, with an unusually low pH optimum of 3.4. The CPn1032 gene complemented a mutation in the Escherichia coli adiA gene, which encodes a pyridoxal 5'-phosphate-dependent arginine decarboxylase, restoring arginine-dependent acid resistance. Acting together with a putative arginine-agmatine antiporter, the CPn1032 homologs may have evolved convergently to form an arginine-dependent acid resistance system. These genes are the first evidence that obligately intracellular chlamydiae may encounter acidic conditions. Alternatively, this system could reduce the host cell arginine concentration and produce inhibitors of nitric oxide synthase.     

Sphingosine-1-phosphate receptor expression in cardiac fibroblasts is modulated by in vitro culture conditions

The bioactive molecule sphingosine-1-phosphate (S1P) binds with high affinity to five recognized receptors (S1P(1-5)) to affect various tissues, including cellular responses of cardiac fibroblasts (CFbs) and myocytes. CFbs are essential components of myocardium, and detailed study of their cell signaling and physiology is required for a number of emerging disciplines. Meaningful studies on CFbs, however, necessitate methods for selective, reproducible cell isolations. Macrophages reside within normal cardiac tissues and often are isolated with CFbs. A protocol was therefore developed that significantly reduces macrophage levels and utilizes more CFb-specific markers (discoidin domain receptor-2) instead of, or in addition to, more commonly used cytoskeletal markers. Our results demonstrate that primary isolated, purified CFbs express predominantly S1P(1-3); however, the relative levels of these receptor subtypes are modulated with time and by culture conditions. In coculture experiments, macrophages altered CFb S1P receptor levels relative to controls. Further investigations using known macrophage-secreted factors showed that S1P and H(2)O(2) had minimal effects on CFb S1P(1-3) expression, whereas transforming growth factor-beta1, TNF-alpha, and PDGF-BB significantly altered all S1P receptor subtypes. Lowering FBS concentrations from 10% to 0.1% increased S1P(2), whereas supplementation with either PDGF-BB or Rho-associated protein kinase inhibitor Y-27632 significantly elevated S1P(3) levels. S1P(2) and S1P(3) receptor levels are known to regulate cell migration. Using cells isolated from either normal or S1P(3)-null mice, we demonstrate that S1P(3) is important and necessary for CFb migration. These results highlight the importance of demonstrating CFb culture purity in functional studies of S1P and also identify conditions that modulate S1P receptor expression in CFbs.     

An analysis of the effects of stretch on IGF-I secretion from rat ventricular fibroblasts

Mechanical force can induce a number of fundamental short- and long-term responses in myocardium. These include alterations in ECM, activation of cell-signaling pathways, altered gene regulation, changes in cell proliferation and growth, and secretion of a number of peptides and growth factors. It is now known that a number of these autocrine/paracrine factors are secreted from both cardiomyocytes and ventricular cardiac fibroblasts (CFb) in response to stretch. One such substance is IGF-I. IGF-I is an important autocrine/paracrine factor that can regulate physiological or pathophysiological responses, such as hypertrophy. In this study, we addressed the possible effects of mechanical perturbation, biaxial strain, on IGF-I secretion from adult rat CFb. CFb were subjected to either static stretch (3-10%) or cyclic stretch (10%; 0.1-1 Hz) over a 24-h period. IGF-1 secretion from CFb in response to selected stretch paradigms was examined using ELISA to measure IGF-I concentrations in conditioned media. Static stretch did not result in any measurable modulation of IGF-I secretion from CFb. However, cyclic stretch significantly increased IGF-I secretion from CFb in a frequency- and time-dependent manner compared with nonstretched controls. This stretch-induced increase in secretion was relatively insensitive to changes in extracellular [Ca(2+)] or to block of L-type Ca(2+) channels. In contrast, thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPase, remarkably decreased stretch-induced IGF-I secretion from CFb. We further show that IGF-I can upregulate mRNA expression of atrial natriuretic peptide in myocytes. In summary, cyclic stretch can significantly increase IGF-I secretion from CFb, and this effect is dependent on a thapsigargin-sensitive pool of intracellular [Ca(2+)].     

Dopamine mediates context-dependent modulation of sensory plasticity in C. elegans

Dopamine has been implicated in the modulation of diverse forms of behavioral plasticity, including appetitive learning and addiction. An important challenge is to understand how dopamine's effects at the cellular level alter the properties of neural circuits to modify behavior. In the nematode C. elegans, dopamine modulates habituation of an escape reflex triggered by body touch. In the absence of food, animals habituate more rapidly than in the presence of food; this contextual information about food availability is provided by dopaminergic mechanosensory neurons that sense the presence of bacteria. We find that dopamine alters habituation kinetics by selectively modulating the touch responses of the anterior-body mechanoreceptors; this modulation involves a D1-like dopamine receptor, a Gq/PLC-beta signaling pathway, and calcium release within the touch neurons. Interestingly, the body touch mechanoreceptors can themselves excite the dopamine neurons, forming a positive feedback loop capable of integrating context and experience to modulate mechanosensory attention.     

Genetically based adaptive trait shifts at an expanding mangrove range margin

Hydrobiologia - Neotropical black mangrove (Avicennia germinans) is expanding poleward into temperate salt marsh along Atlantic Florida, USA, with field evidence of trait shifts consistent with...