Proteomics Analysis of Cancer Exosomes Using a Novel Modified Aptamer-based Array (SOMAscanTM) Platform

We have used a novel affinity-based proteomics technology to examine the protein signature of small secreted extracellular vesicles called exosomes. The technology uses a new class of protein binding reagents called SOMAmers® (slow off-rate modified aptamers) and allows the simultaneous precise measurement of over 1000 proteins. Exosomes were highly purified from the Du145 prostate cancer cell line, by pooling selected fractions from a continuous sucrose gradient (within the density range of 1.1 to 1.2 g/ml), and examined under standard conditions or with additional detergent treatment by the SOMAscan^TM array (version 3.0). Lysates of Du145 cells were also prepared, and the profiles were compared. Housekeeping proteins such as cyclophilin-A, LDH, and Hsp70 were present in exosomes, and we identified almost 100 proteins that were enriched in exosomes relative to cells. These included proteins of known association with cancer exosomes such as MFG-E8, integrins, and MET, and also those less widely reported as exosomally associated, such as ROR1 and ITIH4. Several proteins with no previously known exosomal association were confirmed as exosomally expressed in experiments using individual SOMAmer® reagents or antibodies in micro-plate assays. Western blotting confirmed the SOMAscan^TM-identified enrichment of exosomal NOTCH-3, L1CAM, RAC1, and ADAM9. In conclusion, we describe here over 300 proteins of hitherto unknown association with prostate cancer exosomes and suggest that the SOMAmer®-based assay technology is an effective proteomics platform for exosome-associated biomarker discovery in diverse clinical settings.Prostate carcinoma is the most frequent male cancer, with an estimated 240,000 newly diagnosed individuals and 28,000 deaths in the United States during 2012 (National Cancer Institute (NIH)). Methods for detecting this cancer are based on a combination of physical examination through digital rectal examination, clinical imaging, quantification of circulating levels of prostate specific antigen (PSA),¹ and transrectal ultrasound-guided biopsy. As a non-invasive test, PSA measurement is still widely used, but it remains insensitive, as around 15% of men with normal levels of PSA will have prostate cancer according to biopsy results (1), and 60% of men with elevated PSA levels may have other, noncancerous conditions but be subjected to further, unnecessary investigations and interventions (2). PSA may be of better utility in monitoring disease progression (2). An ability to diagnose the disease more specifically at an early stage is likely to save lives and alleviate the healthcare burden and morbidities arising from misdiagnosis. In addition, methods for monitoring the course of the disease in a non-invasive and perhaps predictive manner would offer increased patient benefit, enabling early detection of imminent relapse under hormone therapy, for example. Therefore there is a clinical need for improved molecular approaches for disease diagnosis and monitoring in these settings.Small vesicles termed exosomes are present in body fluids, including serum, plasma, urine, and seminal plasma (3–7), and their isolation and examiniation may prove useful as a minimally invasive means of obtaining a complex set of disease markers. Exosomes are secreted by most, if not all, cell types and are generally accepted as derived principally from multivesicular bodies of the late endocytic tract (8), although examples of plasma membrane budding nanovesicles of similar phenotype have also been described (9). Exosomes are particularly enriched in membrane proteins and in factors related to such endosomal compartments. They also contain proteins found in the cytosol, but they poorly represent components of organelles such as the mitochondria, nucleus, and endoplasmic reticulum (10). Exosomes also comprise an assortment of coding and noncoding RNA. There has been considerable global effort toward defining disease-related alterations in exosomal RNA. However, it is well established that aberrant alterations in cancer cells in response to metabolic, hypoxic, or other forms of stress are reflected in protein changes in the exosomes produced (11–13). Thus exosomes from diseased origins can be distinguished from those of a normal phenotype based on their protein profiles alone.Proteomics studies using mass spectrometry (MS) have previously been conducted on prostate cancer exosomes/microvesicles obtained from cell lines (14, 15), xenotransplantation models (16), or ex vivo biofluids (17). Hundreds of proteins with putative associations with exosomes/microvesicles have been identified. These studies highlight several interesting candidate markers of potential biomarker utility that are currently being explored. However, global proteomic approaches of this nature can have two major limitations. Although the most abundant proteins are more likely to be identified by MS, it is difficult to infer information about relative abundances of proteins in complex samples when using these methods. Secondly, given the often exacting, difficult-to-reproduce, and time-consuming workflows involved, such technologies are poorly suited for the analysis of a large number of samples. Multiplex protein array methodologies have the potential to overcome such issues and offer quantification and options for more rapid sample throughput. However, most platforms are based on antibodies, and these arrays are typically limited to <100 proteins, principally because the cross-reactivity of secondary antibodies can negatively affect assay specificity (18).A recently developed proteomics platform, termed SOMAscan^TM, provides a new generation of protein detection technologies. The platform is capable of the simultaneous quantitative analysis of 1129 proteins per sample in its current form. It is also an approach well suited to handling large numbers of specimens required for well-powered clinical studies (19). The key to this technology, which is described in detail by Gold et al. (20, 21), is the use of slow off-rate modified aptamers (SOMAmers) containing chemically modified nucleotides. This confers greater stability, expanded target range, and improved affinity for the target proteins. This multiplex platform has been applied successfully to small volumes (∼15 μl) of plasma specimens from chronic renal disease patients (20), serum specimens from mesothelioma (22) or lung cancer patients (19), tissue lysates (23), and cerebrospinal fluid (24). However, to date, the compatibility of this array technology with exosomes as the specimen has not been investigated.The purpose of the current study was to examine the utility of this evolving technology in profiling the protein repertoire of exosomes. Research was conducted using highly pure exosomes isolated from a prostate cancer cell line, and we compared this sample to the protein profile of the parent cells. By so doing, we obtained evidence of the compatibility of the platform with this difficult, membranous sample and identified several proteins of previously unknown association with exosomes. In summary, SOMAscan^TM is a versatile tool for probing the composition of exosomes and is a suitable platform to provide a high-throughput approach for exosome-based biomarker discovery in prostate cancer and other clinical settings.     

Establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation in banana cv. ‘Dwarf Cavendish’ (Musa AAA): effect of spermidine on transformation efficiency

An efficient method has been developed for somatic embryogenesis, plant regeneration and transformation of the important banana cultivar ‘Dwarf Cavendish’ (Musa AAA). A high embryogenic response was obtained in 1.36 % of immature male flower explants. Once embryogenic structures were transferred to liquid medium, embryogenic cell suspensions (ECSs) with high regeneration capacity were obtained. ECSs were incubated under different conditions with Agrobacterium tumefaciens strain EHA101 harboring vector pFAJ3000 that contains pNos-nptII-tOcs and p35S-uidAintron-t35S expression cassettes. The effect of spermidine and infection time on transformation efficiency was examined. The highest efficiency was obtained when ECSs were infected for 6 h, in medium supplemented with 200 μM acetosyringone and 1.0 mM spermidine, with more than 600 independent lines/~50 mg FW of settled cells. Spermidine showed an enhancing effect, increasing significantly the transient Gus expression and the number of transformed embryo colonies and regenerated plants in comparison with the same treatments without this polyamine. This is the first report showing efficient Agrobacterium tumefaciens mediated transformation using embryogenic cell suspension cultures in the ‘Dwarf Cavendish’ banana cultivar.     

The role of passive calcium influx through the cell membrane in galvanotaxis

Passive calcium influx is one of the theories to explain the cathodal galvanotaxis of cells that utilize the electric field to guide their motion. When exposed to an electric field, the intracellular fluid becomes polarized, leading to positive charge accumulation on the cathodal side and negative charge accumulation on the anodal side. The negative charge on the anodal side attracts extracellular calcium ions, increasing the anodal calcium concentration, which is supposed to decrease the mobile properties of this side. Unfortunately, this model does not capture the Ca²⁺ dynamics after its presentation to the intracellular fluid. The ions cannot permanently accumulate on the anodal side because that would build a potential drop across the cytoplasm leading to an ionic current, which would carry positive ions (not only Ca²⁺) from the anodal to the cathodal part through the cytoplasm. If the cytoplasmic conductance for Ca²⁺ is low enough compared to the membrane conductance, the theory could correctly predict the actual behavior. If the ions move through the cytoplasm at a faster rate, compensating for the passive influx, this theory may fail. This paper contains a discussion of the regimes of validity for this theory.     

Gravity is a pull! Exploring forces and motion with English learners

English learners (ELs) benefit from inquiry-based science instruction that includes explicit attention to language learning goals. The purpose of this article is to share a third-grade unit on forces and motion which integrates science inquiry and writing in science notebooks with the goal of developing ELs' engagement in science, conceptual understanding, and academic language and literacy skills. We demonstrate how to engage diverse students' background knowledge and use classroom activities and discussion to create bridges between everyday and academic language. We utilize excerpts from Peter, Lucia, and Andrea's science notebooks to explore and highlight how teachers can use this resource as a means of communicating science, during instruction. Through these EL students' journals, we discuss the importance of developing language goals at the word, sentence, and discourse level while promoting and supporting ELs' use of the language of science.     

The presence of two major protein components in the bovine photoreceptor disc membrane.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of thoroughly washed rod outer segment membrane preparations from bovine retinae revealed two major membrane-bound components and not one as has been generally thought. The higher molecular weight peak (?38500 molecular weight) contains a carbohydrate component and is covalently bound to the retinylidene chromophore. Moreover, this material is extensively phosphorylated $\text{in vitro}$ upon illumination. Therefore, this component (peak H) is rhodopsin. The nature and function of the other photoreceptor disc membrane component (peak L, ?34500 molecular weight) remains to be determined.     

Body height in relation to rural-urban migration in Poland

A survey was conducted among 2800 students studying in Wroc?aw, Poland. The questionnaire included questions on the body height of the students and their parents, and place of residence and migration patterns of the students themselves, their parents and their grandparents. Body height in both students and their parents was positively correlated with the size of their place of residence. This was particularly true for male students and their fathers. Body height in students and parents from mobile families was not significantly different from that of their peers from non-mobile families. Body height in mobile individuals was generally between that of non-mobile individuals from rural areas and that of non-mobile individuals from large urban centres. Students from families that had migrated from smaller urban centres to larger ones were taller than students from families that had migrated from rural areas to urban centres. Body height in students was also correlated with the kind of migration that took place. In the students' mothers, body height was higher if the maternal grandparents moved from smaller urban centres to larger urban centres than if the maternal grandparents moved from rural areas to urban centres. In female students, body height depended on whether their mothers had migrated from smaller places of residence to larger places of residence, but was not affected by the degree of migration. Intra-generational migration during the generation of the students' grandparents was associated with increased body height in the students' mothers. On the other hand, intergenerational migration during the generations of the students' grandparents and parents was associated with increased body height in the students' fathers and in female students. Body height was not a reliable indicator of whether an individual migrated from rural areas to Wroc?aw. Far more reliable indicators were the size of the place the student lived their whole life and whether the family had lived in an urban environment for at least two generations.     

Distinct timing mechanisms produce discrete and continuous movements

The differentiation of discrete and continuous movement is one of the pillars of motor behavior classification. Discrete movements have a definite beginning and end, whereas continuous movements do not have such discriminable end points. In the past decade there has been vigorous debate whether this classification implies different control processes. This debate up until the present has been empirically based. Here, we present an unambiguous non-empirical classification based on theorems in dynamical system theory that sets discrete and continuous movements apart. Through computational simulations of representative modes of each class and topological analysis of the flow in state space, we show that distinct control mechanisms underwrite discrete and fast rhythmic movements. In particular, we demonstrate that discrete movements require a time keeper while fast rhythmic movements do not. We validate our computational findings experimentally using a behavioral paradigm in which human participants performed finger flexion-extension movements at various movement paces and under different instructions. Our results demonstrate that the human motor system employs different timing control mechanisms (presumably via differential recruitment of neural subsystems) to accomplish varying behavioral functions such as speed constraints.     

Striking ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all four human,and the Drosophila melanogaster,deoxyribonucleoside kinases

In extension of an earlier report, six non-conventional analogues of ATP, three adenosine-2'-triphosphates (3'-deoxy, 3'-deoxy-3'-fluoro- and 3'-deoxy-3'-fluoroxylo-), and three adenosine-3'-triphosphates (2'-deoxy-, 2'-deoxy-2'-fluoro- and 2'-deoxy-2'-fluoroara-), were compared with ATP as potential phosphate donors for human deoxycytidine kinase (dCK), cytosolic thymidine kinase (TK1), mitochondrial TK2, deoxyguanosine kinase (dGK), and the deoxyribonucleoside kinase (dNK) from Drosophila melanogaster. With one group of enzymes, comprising TK1, TK2, dNK and dCK (with dAdo as acceptor), only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP), and the other five analogues much less so, or inactive. With a second set, including dCK (dCyd, but not dAdo, as acceptor) and dGK (dGuo as acceptor), known to share high sequence similarity (approximately 45% sequence identity), all six analogues were good to excellent donors (13-119% that for ATP). With dCK and ATP1, products were shown to be 5'-phosphates. With dCK, donor properties of the analogues were dependent on the nature of the acceptor, as with natural 5'-triphosphate donors. With dCK (dCyd as acceptor), Km and Vmax for the two 2'(3')-deoxyadenosine-3'(2')-triphosphates are similar to those for ATP. With dGK, Km values are higher than for ATP, while Vmax values are comparable. Kinetic studies further demonstrated Michaelis-Menten (non-cooperative) or cooperative kinetics, dependent on the enzyme employed and the nature of the donor. The physiological significance, if any, of the foregoing remains to be elucidated. The overall results are, on the other hand, highly relevant to studies on the modes of interaction of nucleoside kinases with donors and acceptors; and, in particular, to interpretations of the recently reported crystal structures of dGK with bound ATP, of dNK with bound dCyd, and associated modeling studies.