Bone sialoprotein supports breast cancer cell adhesion proliferation and migration through differential usage of the αvβ3 and αvβ5 integrins

Bone sialoprotein (BSP), a secreted glycoprotein found in bone matrix, has been implicated in the formation of mammary microcalcifications and osteotropic metastasis of human breast cancer (HBC). BSP possesses an integrin-binding RGD (Arg-Gly-Asp) domain, which may promote interactions between HBC cells and bone extracellular matrix. Purified BSP, recombinant human BSP fragments and BSP-derived RGD peptides are shown to elicit migratory, adhesive, and proliferative responses in the MDA-MB-231 HBC cell line. Recombinant BSP fragment analysis localized a significant component of these activities to the RGD domain of the protein, and synthetic RGD peptides with BSP flanking sequences (BSPRGD) also conferred these responses. The fibronectin-derived RGD counterpart, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), could not support these cellular responses, emphasizing specificity of the BSP configuration. Although most of the proliferative and adhesive responses could be attributed to RGD interactions, these interactions were only partly responsible for the migrational responses. Experiments with integrin-blocking antibodies demonstrated that BSP-RGD-induced migration utilizes the αvβ3 vitronectin receptor, whereas adhesion and proliferation responses were αvβ5-mediated. Using fluorescence activated cell sorting, we selected two separate subpopulations of MDA-MB-231 cells enriched for αvβ3 or αvβ5 respectively. Although some expression of the alternate αv integrin was still retained, the αvβ5-enriched MDA-MB-231 cells showed enhanced proliferative and adhesive responses, whereas the αvβ3-enriched subpopulation was suppressed for proliferation and adhesion, but showed enhanced migratory responses to BSP-RGD. In addition, similar analysis of two other HBC cell lines showed less marked, but similar RGD-dependent trends in adhesion and proliferation to the BSP fragments. Collectively, these data demonstrate BSP effects on proliferative, migratory, and adhesive functions in HBC cells and that the RGD-mediated component differentially employs αvβ3 and αvβ5 integrin receptors.

Models for studying cellular invasion of basement membranes

Invasion of extracellular matrices is crucial to a number of physiological and pathophysiological states, including tumor cell metastasis, arthritis, embryo implantation, wound healing, and early development. To isolate invasion from the additional complexities of these scenarios a number of in vitro invasion assays have been developed over the years. Early studies employed intact tissues, like denuded amniotic membrane (1) or embryonic chick heart fragments (2), however recently, purified matrix components or complex matrix extracts have been used to provide more uniform and often more rapid analyses (for examples, see the following integrin studies). Of course, the more holistic view of invasion offered in the earlier assays is valuable and cannot be fully reproduced in these more rapid assays, but advantages of reproducibility among replicates, ease of preparation and analysis, and overall high throughput favor the newer assays. In this chapter, we will focus on providing detailed protocols for Matrigel-based assays (Matrigel=reconstituted basement membrane; reviewed in ref. (3)). Matrigel is an extract from the transplantable Engelbreth-Holm-Swarm murine sarcoma that deposits a multilammelar basement membrane. Matrigel is available commercially (Becton Dickinson, Bedford, MA), and can be manipulated as a liquid at 4°C into a variety of different formats. Alternatively, cell culture inserts precoated with Matrigel can be purchased for even greater simplicity.

Analysis of albumin-associated peptides and proteins from ovarian cancer patients

Albumin binds low–molecular-weight molecules, including proteins and peptides, which then acquire its longer half-life, thereby protecting the bound species from kidney clearance. We developed an experimental method to isolate albumin in its native state and to then identify [mass spectrometry (MS) sequencing] the corresponding bound low–molecular-weight molecules. We used this method to analyze pooled sera from a human disease study set (high-risk persons without cancer, n= 40; stage I ovarian cancer, n = 30; stage III ovarian cancer, n = 40) to demonstrate the feasibility of this approach as a discovery method. Methods Albumin was isolated by solid-phase affinity capture under native binding and washing conditions. Captured albumin-associated proteins and peptides were separated by gel electrophoresis and subjected to iterative MS sequencing by microcapillary reversed-phase tandem MS. Selected albumin-bound protein fragments were confirmed in human sera by Western blotting and immunocompetition. Results In total, 1208 individual protein sequences were predicted from all 3 pools. The predicted sequences were largely fragments derived from proteins with diverse biological functions. More than one third of these fragments were identified by multiple peptide sequences, and more than one half of the identified species were in vivo cleavage products of parent proteins. An estimated 700 serum peptides or proteins were predicted that had not been reported in previous serum databases. Several proteolytic fragments of larger molecules that may be cancer-related were confirmed immunologically in blood by Western blotting and peptide immunocompetition. BRCA2, a 390-kDa low-abundance nuclear protein linked to cancer susceptibility, was represented in sera as a series of specific fragments bound to albumin. Conclusion Carrier-protein harvesting provides a rich source of candidate peptides and proteins with potential diverse tissue and cellular origins that may reflect important disease-related information.

Plasma-deposited Ge nanoisland films on Si : is Stranski–Krastanow fragmentation unavoidable?

The formation of Ge quantum dot arrays by deposition from a low-temperature plasma environment is investigated by kinetic Monte Carlo numerical simulation. It is demonstrated that balancing of the Ge influx from the plasma against surface diffusion provides an effective control of the surface processes and can result in the formation of very small densely packed quantum dots. In the supply-controlled mode, a continuous layer is formed which is then followed by the usual Stranski-Krastanow fragmentation with a nanocluster size of 10 nm. In the diffusion-controlled mode, with the oversupply relative to the surface diffusion rate, nanoclusters with a characteristic size of 3 nm are formed. Higher temperatures change the mode to supply controlled and thus encourage formation of the continuous layer that then fragments into an array of large size. The use of a high rate of deposition, easily accessible using plasma techniques, changes the mode to diffusion controlled and thus encourages formation of a dense array of small nanoislands.

The amplified peptidome : the new treasure chest of candidate biomarkers

Mass spectrometric analysis of the low-molecular weight (LMW) range of the serum/plasma proteome is revealing the existence of large numbers of previously unknown peptides and protein fragments predicted to be derived from low- abundance proteins. This raises the question of why such low abundance molecules would be retained at detectable levels in the circulation, instead of being rapidly cleared and excreted. Theoretical models of biomarker production and association with serum carrier proteins have been developed to elucidate the mechanisms governing biomarker half-life in the bloodstream. These models predict that the vast majority of LMW biomarkers exist in association with circulating high molecular mass carrier proteins. Moreover, the total serum/ plasma concentration of the biomarker is largely determined by the clearance rate of the carrier protein, not the free-phase biomarker clearance itself. These predictions have been verified experimentally using molecular mass fractionation of human serum before mass spectrometry sequence analysis. These principles have profound implications for biomarker discovery and measurement.

The crude skin secretion of the pepper frog leptodactylus labyrinthicus is rich in metallo and serine peptidases

Peptidases are ubiquitous enzymes involved in diverse biological processes. Fragments from bioactive peptides have been found in skin secretions from frogs, and their presence suggests processing by peptidases. Thus, the aim of this work was to characterize the peptidase activity present in the skin secretion of Leptodactylus labyrinthicus. Zymography revealed the presence of three bands of gelatinase activity of approximately 60 kDa, 66 kDa, and 80 kDa, which the first two were calcium-dependent. These three bands were inhibited either by ethylenediaminetetraacetic acid (EDTA) and phenathroline; thus, they were characterized as metallopeptidases. Furthermore, the proteolytic enzymes identified were active only at pH 6.0–10.0, and their activity increased in the presence of CHAPS or NaCl. Experiments with fluorogenic substrates incubated with skin secretions identified aminopeptidase activity, with cleavage after leucine, proline, and alanine residues. This activity was directly proportional to the protein concentration, and it was inhibited in the presence of metallo and serine peptidase inhibitors. Besides, the optimal pH for substrate cleavage was determined to be 7.0–8.0. The results of the in gel activity assay showed that all substrates were hydrolyzed by a 45 kDa peptidase. Gly-Pro-AMC was also cleaved by a peptidase greater than 97 kDa. The data suggest the presence of dipeptidyl peptidases (DPPs) and metallopeptidases; however, further research is necessary. In conclusion, our work will help to elucidate the implication of these enzymatic activities in the processing of the bioactive peptides present in frog venom, expanding the knowledge of amphibian biology.

Independent functions of yeast Pcf11p in pre-mRNA 3' end processing and in transcription termination

Pcf11p, an essential subunit of the yeast cleavage factor IA, is required for pre‐mRNA 3′ end processing, binds to the C‐terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) and is involved in transcription termination. We show that the conserved CTD interaction domain (CID) of Pcf11p is essential for cell viability. Interestingly, the CTD binding and 3′ end processing activities of Pcf11p can be functionally uncoupled from each other and provided by distinct Pcf11p fragments in trans. Impaired CTD binding did not affect the 3′ end processing activity of Pcf11p and a deficiency of Pcf11p in 3′ end processing did not prevent CTD binding. Transcriptional run‐on analysis with the CYC1 gene revealed that loss of cleavage activity did not correlate with a defect in transcription termination, whereas loss of CTD binding did. We conclude that Pcf11p is a bifunctional protein and that transcript cleavage is not an obligatory step prior to RNAP II termination.

“My hands are plunged in oranges”…: an experiential account of a working process

This presentation incorporated the live performance throughout, by the author, of movement from “The All Weather Project” by Liz Roche. Movement sections are indicated by italics. “I am going to start by dancing for you… Movement: Live performance of solo approximately 10 minutes in duration This is the introduction... Through my PhD research, I am examining the choreographic process from the perspective of the independent contemporary dancer, through embodying this role as a researcher/participant. My methodological frameworks, which utilise video documentation and journal writing, could be characterised as ethnographic, multi-modal embodied theorising, leading to “multi-dimensional theorising” (I adopt this term from Susan Melrose). In this way, I am unwinding the embodied practice of dancing, through the co-existent layers of experience, towards forming a theoretical understanding of the issues that arise for the dancer. The issues that I have identified as relevant to my research are those relating to the dancer’s ‘moving identity’ or way of moving, as a mutable and adaptable form that must alter and re-adjust to each different choreographic engram or movement vocabulary, that she/he encounters. I am examining this interplay between stability and change. I also reflect on the impact of destabilisation and flux on the dancer’s identity in a wider sense, as she/he relates outwardly to signifying factors within the social strata. Today I am going to bring you through a reflection on the working process of a dance piece as experienced from the inside. By doing so, I hope to capture and elucidate the multi-dimensional layers which existed for me within this process. Through displaying these fragments together, I endeavour to invoke the ‘totality’ of the experience...

Between rocks and hard places

A travel article about the Aland Islands, Finland, that discusses the mix of Swedish, Finnish, and Russian cultural influences in the area. On the map, Finland seems to end in fragments. The gods have stomped their heels on the southwestern corner, and between the cities of Helsinki and Turku it is jagged, rocky islands that form the final landmarks...

A rapid and cost-effective method of producing recombinant proBNP and NT-proBNP variants in Escherichia coli for immunoassay of heart failure

The measurements of plasma natriuretic peptides (NT-proBNP, proBNP and BNP) are used to diagnose heart failure but these are expensive to produce. We describe a rapid, cheap and facile production of proteins for immunoassays of heart failure. DNA encoding N-terminally His-tagged NT-proBNP and proBNP were cloned into the pJexpress404 vector. ProBNP and NT-proBNP peptides were expressed in Escherichia coli, purified and refolded in vitro. The analytical performance of these peptides were comparable with commercial analytes (NT-proBNP EC50 for the recombinant is 2.6 ng/ml and for the commercial material is 5.3 ng/ml) and the EC50 for recombinant and commercial proBNP, are 3.6 and 5.7 ng/ml respectively). Total yield of purified refolded NT-proBNP peptide was 1.75 mg/l and proBNP was 0.088 mg/l. This approach may also be useful in expressing other protein analytes for immunoassay applications. To develop a cost effective protein expression method in E. coli to obtain high yields of NT-proBNP (1.75 mg/l) and proBNP (0.088 mg/l) peptides for immunoassay use.

Detecting approximate clones in business process model repositories

Empirical evidence shows that repositories of business process models used in industrial practice contain significant amounts of duplication. This duplication arises for example when the repository covers multiple variants of the same processes or due to copy-pasting. Previous work has addressed the problem of efficiently retrieving exact clones that can be refactored into shared subprocess models. This article studies the broader problem of approximate clone detection in process models. The article proposes techniques for detecting clusters of approximate clones based on two well-known clustering algorithms: DBSCAN and Hi- erarchical Agglomerative Clustering (HAC). The article also defines a measure of standardizability of an approximate clone cluster, meaning the potential benefit of replacing the approximate clones with a single standardized subprocess. Experiments show that both techniques, in conjunction with the proposed standardizability measure, accurately retrieve clusters of approximate clones that originate from copy-pasting followed by independent modifications to the copied fragments. Additional experiments show that both techniques produce clusters that match those produced by human subjects and that are perceived to be standardizable.

Temporal and spatial distribution and habitat associations of an urban population of New Zealand long-tailed bats (Chalinolobus tuberculatus)

Two ultrasound survey methods were used to determine the presence and activity patterns of New Zealand long-tailed bats (Chalinolobus tuberculatus) in the city of Hamilton. First, 13 monthly surveys conducted at 18 green spaces found C. tuberculatus in only one urban forest reserve, Hammond Bush, where they were found consistently throughout the year. Bat activity was strongly related to temperature. Second, twice-yearly citywide surveys conducted over 2 years determined the distribution and habitat associations of C. tuberculatus. Bats were found only in the southern part of the city and were strongly associated with the Waikato River. Bat activity was negatively correlated with housing and street light density and positively correlated with topographical complexity. In Hamilton, topographical complexity indicates the presence of gullies. Gullies probably provide foraging and roosting opportunities and connect the river to distant forest patches. These results suggest that urban habitats can be useful for bats if gullies can link these to distant habitat fragments.

Establishing prostate cancer patient derived xenografts: Lessons learned from older studies

Background Understanding the progression of prostate cancer to androgen-independence/castrate resistance and development of preclinical testing models are important for developing new prostate cancer therapies. This report describes studies performed 30 years ago, which demonstrate utility and shortfalls of xenografting to preclinical modeling. Methods We subcutaneously implanted male nude mice with small prostate cancer fragments from transurethral resection of the prostate (TURP) from 29 patients. Successful xenografts were passaged into new host mice. They were characterized using histology, immunohistochemistry for marker expression, flow cytometry for ploidy status, and in some cases by electron microscopy and response to testosterone. Two xenografts were karyotyped by G-banding. Results Tissues from 3/29 donors (10%) gave rise to xenografts that were successfully serially passaged in vivo. Two, (UCRU-PR-1, which subsequently was replaced by a mouse fibrosarcoma, and UCRU-PR-2, which combined epithelial and neuroendocrine features) have been described. UCRU-PR-4 line was a poorly differentiated prostatic adenocarcinoma derived from a patient who had undergone estrogen therapy and bilateral castration after his cancer relapsed. Histologically, this comprised diffusely infiltrating small acinar cell carcinoma with more solid aggregates of poorly differentiated adenocarcinoma. The xenografted line showed histology consistent with a poorly differentiated adenocarcinoma and stained positively for prostatic acid phosphatase (PAcP), epithelial membrane antigen (EMA) and the cytokeratin cocktail, CAM5.2, with weak staining for prostate specific antigen (PSA). The line failed to grow in female nude mice. Castration of three male nude mice after xenograft establishment resulted in cessation of growth in one, growth regression in another and transient growth in another, suggesting that some cells had retained androgen sensitivity. The karyotype (from passage 1) was 43–46, XY, dic(1;12)(p11;p11), der(3)t(3:?5)(q13;q13), -5, inv(7)(p15q35) x2, +add(7)(p13), add(8)(p22), add(11)(p14), add(13)(p11), add(20)(p12), -22, +r4[cp8]. Conclusions Xenografts provide a clinically relevant model of prostate cancer, although establishing serially transplantable prostate cancer patient derived xenografts is challenging and requires rigorous characterization and high quality starting material. Xenografting from advanced prostate cancer is more likely to succeed, as xenografting from well differentiated, localized disease has not been achieved in our experience. Strong translational correlations can be demonstrated between the clinical disease state and the xenograft model

Is there a bone-nail specific entry point? automated fit quantification of tibial nail designs during the insertion for six different nail entry points

Intramedullary nailing is the standard fixation method for displaced diaphyseal fractures of tibia. Selection of the correct nail insertion point is important for axial alignment of bone fragments and to avoid iatrogenic fractures. However, the standard entry point (SEP) may not always optimise the bone-nail fit due to geometric variations of bones. This study aimed to investigate the optimal entry for a given bone-nail pair using the fit quantification software tool previously developed by the authors. The misfit was quantified for 20 bones with two nail designs (ETN and ETN-Proximal Bend) related to the SEP and 5 entry points which were 5 mm and 10 mm away from the SEP. The SEP was the optimal entry point for 50% of the bones used. For the remaining bones, the optimal entry point was located 5 mm away from the SEP, which improved the overall fit by 40% on average. However, entry points 10 mm away from the SEP doubled the misfit. The optimised bone-nail fit can be achieved through the SEP and within the range of a 5 mm radius, except posteriorly. The study results suggest that the optimal entry point should be selected by considering the fit during insertion and not only at the final position.

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction.