A copia-like element in Pisum demonstrates the uses of dispersed repeated sequences in genetic analysis

A DNA sequence between two legumin genes in Pisum is a member of the copia-like class of retrotransposons and represents one member of a polymorphic and heterogeneous dispersed repeated sequence family in Pisum. This sequence can be exploited in genetic studies either by RFLP analysis where several markers can be scored together, or the segregation of individual elements can be followed after PCR amplification of specific members.

Ozone-Induced Dissociation on a Modified Tandem Linear Ion-Trap : Observations of Different Reactivity for Isomeric Lipids

Ozone-induced dissociation (OzID) exploits the gas-phase reaction between mass-selected lipid ions and ozone vapor to determine the position(s) of unsaturation In this contribution, we describe the modification of a tandem linear ion-trap mass spectrometer specifically for OzID analyses wherein ozone vapor is supplied to the collision cell This instrumental configuration provides spatial separation between mass-selection, the ozonolysis reaction, and mass-analysis steps in the OzID process and thus delivers significant enhancements in speed and sensitivity (ca 30-fold) These improvements allow spectra revealing the double-bond position(s) within unsaturated lipids to be acquired within 1 s significantly enhancing the utility of OzID in high-throughput lipidomic protocols The stable ozone concentration afforded by this modified instrument also allows direct comparison of relative reactivity of isomeric lipids and reveals reactivity trends related to (1) double-bond position, (2) substitution position on the glycerol backbone, and (3) stereochemistry For cis- and trans-isomers, differences were also observed in the branching ratio of product ions arising from the gas-phase ozonolysis reaction, suggesting that relative ion abundances could be exploited as markers for double-bond geometry Additional activation energy applied to mass-selected lipid ions during injection into the collision cell (with ozone present) was found to yield spectra containing both OzID and classical-CID fragment ions This combination CID-OzID acquisition on an ostensibly simple monounsaturated phosphatidylcholine within a cow brain lipid extract provided evidence for up to four structurally distinct phospholipids differing in both double-bond position and sn-substitution U Am Soc Mass Spectrom 2010, 21, 1989-1999) (C) 2010 American Society for Mass Spectrometry

Loss of epithelial markers and acquisition of vimentin expression in adriamycin- and vinblastine-resistant human breast cancer cell lines

We have previously observed that breast cancer cell lines could exhibit either epithelial or fibroblastic phenotypes as reflected by their morphologies and intermediate filament protein expression (C. L. Sommers, D. Walker-Jones, S. E. Heckford, P. Worland, E. Valverius, R. Clark, M. Stampfer, and E. P. Gelmann, Cancer Res., 49: 4258-4263, 1989). Fibroblastoid, vimentin-expressing breast cancer cell lines are more invasive in vitro and in vivo (E. W. Thompson, S. Paik, N. Brunner, C. L. Sommers, G. Zugmaier, R. Clarke, T. B. Shima, J. Torri, S. Donahue, M. E. Lippman, G. R. Martin, and R. B. Dickson, J. Cell. Physiol., 150: 534-544, 1992). We hypothesized that a breast cancer cell with an epithelial phenotype could undergo a transition to a fibroblastic phenotype, possibly resulting in more invasive capacity. We now show that two Adriamycin-resistant MCF-7 cell lines and a vinblastine-resistant ZR-75-B cell line have undergone such a transition. Adriamycin-resistant MCF-7 cells express vimentin, have diminished keratin 19 expression, have lost cell adhesion molecule uvomorulin expression, and have reduced formation of desmosomes and tight junctions as determined by reduced immunodetection of their components desmoplakins I and II and zonula occludens (ZO)-1. Other MCF-7 cell lines selected for resistance to vinblastine and to Adriamycin and verapamil did not have these characteristics, indicating that drug selection does not invariably cause these phenotypic changes. In addition, to determine if vimentin expression in MCF-7 cells alone could manifest a fibroblastic phenotype, we transfected the full-length human vimentin complementary DNA into MCF-7 cells. Although vimentin expression was achieved in MCF-7 cells, it did not affect the phenotype of the cells in terms of the distribution of keratins, desmoplakins I and II, ZO-1, or uvomorulin or in terms of in vitro invasiveness. We conclude that vimentin expression is a marker for a fibroblastic and invasive phenotype in breast cancer cells but does not by itself give rise to this phenotype.

Paracrine interactions between LNCaP prostate cancer cells and bioengineered bone in 3D in vitro culture reflect molecular changes during bone metastasis

As microenvironmental factors such as three-dimensionality and cell–matrix interactions are increasingly being acknowledged by cancer biologists, more complex 3D in vitro models are being developed to study tumorigenesis and cancer progression. To better understand the pathophysiology of bone metastasis, we have established and validated a 3D indirect co-culture model to investigate the paracrine interactions between prostate cancer (PCa) cells and human osteoblasts. Co-culture of the human PCa, LNCaP cells embedded within polyethylene glycol hydrogels with human osteoblasts in the form of a tissue engineered bone construct (TEB), resulted in reduced proliferation of LNCaP cells. LNCaP cells in both monoculture and co-culture were responsive to the androgen analog, R1881, as indicated by an increase in the expression (mRNA and/or protein induction) of androgen-regulated genes including prostate specific antigen and fatty acid synthase. Microarray gene expression analysis further revealed an up-regulation of bone markers and other genes associated with skeletal and vasculature development and a significant activation of transforming growth factor β1 downstream genes in LNCaP cells after co-culture with TEB. LNCaP cells co-cultured with TEB also unexpectedly showed similar changes in classical androgen-responsive genes under androgen-deprived conditions not seen in LNCaP monocultures. The molecular changes of LNCaP cells after co-culturing with TEBs suggest that osteoblasts exert a paracrine effect that may promote osteomimicry and modulate the expression of androgen-responsive genes in LNCaP cells. Taken together, we have presented a novel 3D in vitro model that allows the study of cellular and molecular changes occurring in PCa cells and osteoblasts that are relevant to metastatic colonization of bone. This unique in vitro model could also facilitate cancer biologists to dissect specific biological hypotheses via extensive genomic or proteomic assessments to further our understanding of the PCa-bone crosstalk.

A light and electron microscopic study of NADPH-diaphorase-,calretinin- and parvalbumin-containing neurons in the rat nucleus accumbens

The rat nucleus accumbens contains medium-sized, spiny projection neurons and intrinsic, local circuit neurons, or interneurons. Sub-classes of interneurons, revealed by calretinin (CR) or parvalbumin (PV) immunoreactivity or reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, were compared in the nucleus accumbens core, shell and rostral pole. CR, PV and NADPH-diaphorase-containing neurons are shown to form three non-co-localising populations in these three areas. No significant differences in neuronal population densities were found between the subterritories. NADPH-diaphorase-containing neurons could be further separated morphologically into three sub-groups, but CR- and PV-immunoreactive neurons form homogeneous populations. Ultrastructurally, NADPH-diaphorase-, CR- and PV-containing neurons in the nucleus accumbens all possess nuclear indentations. These are deeper and fewer in neurons immunoreactive for PV than in CR- and NADPH-diaphorase-containing neurons. CR-immunoreactive boutons form asymmetrical and symmetrical synaptic specialisations on spines, dendrites and somata, while PV-immunoreactive boutons make only symmetrical synaptic specialisations. Both CR- and PV-immunoreactive boutons form symmetrical synaptic specialisations with medium-sized spiny neurons and contact other CR- and PV-immunoreactive somata, respectively. A novel non-carcinogenic substrate for the peroxidase reaction (Vector Slate Grey, SG) was found to be characteristically electron-dense and may be distinguishable from the diaminobenzidine reaction product. We conclude that the three markers used in this study are localised in distinct populations of nucleus accumbens interneurons. Our studies of their synaptic connections contribute to an increased understanding of the intrinsic circuitry of this area.

Beyond tasks and gateways: discovering BPMN models with subprocesses, boundary events and activity markers

Existing techniques for automated discovery of process models from event logs largely focus on extracting flat process models. In other words, they fail to exploit the notion of subprocess, as well as structured error handling and repetition constructs provided by contemporary process modeling notations, such as the Business Process Model and Notation (BPMN). This paper presents a technique for automated discovery of BPMN models containing subprocesses, interrupting and non-interrupting boundary events, and loop and multi-instance markers. The technique analyzes dependencies between data attributes associated with events, in order to identify subprocesses and to extract their associated logs. Parent process and subprocess models are then discovered separately using existing techniques for flat process model discovery. Finally, the resulting models and logs are heuristically analyzed in order to identify boundary events and markers. A validation with one synthetic and two real-life logs shows that process models derived using the proposed technique are more accurate and less complex than those derived with flat process model discovery techniques.

Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins α-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, α-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

The role of biological extracellular matrix scaffolds in vascularized three-dimensional tissue growth in vivo

An in vivo murine vascularized chamber model has been shown to generate spontaneous angiogenesis and new tissue formation. This experiment aimed to assess the effects of common biological scaffolds on tissue growth in this model. Either laminin-1, type I collagen, fibrin glue, hyaluronan, or sea sponge was inserted into silicone chambers containing the epigastric artery and vein, one end was sealed with adipose tissue and the other with bone wax, then incubated subcutaneously. After 2, 4, or 6 weeks, tissue from chambers containing collagen I, fibrin glue, hyaluronan, or no added scaffold (control) had small amounts of vascularized connective tissue. Chambers containing sea sponge had moderate connective tissue growth together with a mild "foreign body" inflammatory response. Chambers containing laminin-1, at a concentration 10-fold lower than its concentration in Matrigel™, resulted in a moderate adipogenic response. In summary, (1) biological hydrogels are resorbed and gradually replaced by vascularized connective tissue; (2) sponge-like matrices with large pores support connective tissue growth within the pores and become encapsulated with granulation tissue; (3) laminin-containing scaffolds facilitate adipogenesis. It is concluded that the nature and chemical composition of the scaffold exerts a significant influence on the amount and type of tissue generated in this in vivo chamber model.

Second-harmonic generation from biological tissues : effect of excitation wavelength

We report on the measurement of second-harmonic signals from hyperplastic parenchyma and stroma in malignant human prostate tissue under femtosecond pulsed illumination in the wavelength range from 730 to 870 nm. In particular, the relationship of the second-harmonic generation to the excitation wavelength is measured. The result in these two regions behaves considerably differently and thus provides a possible indicator for identifying tissue components and malignancy.

Two-photon fluorescence spectroscopy for identification of healthy and malignant biological tissues

Two-photon fluorescence spectroscopy has been performed on rat skeletal muscles to investigate the effect of fixation processes on the micro-environments of the endogenous fluorophors in rat skeletal muscles. The two-photon fluorescence spectra measured for different fixation periods show a differential among those samples that were fixed in water, formalin and methanol, respectively. The results imply that two-photon fluorescence spectroscopy can be a potential technique for identification of healthy and malignant biological tissues.

Biological performance of a polycaprolactone-based scaffold plus recombinant human morphogenetic protein-2 (rhBMP-2) in an ovine thoracic interbody fusion model

PURPOSE. We develop a sheep thoracic spine interbody fusion model to study the suitability of polycaprolactone-based scaffold and recombinant human bone morphogenetic protein-2 (rhBMP-2) as a bone graft substitute within the thoracic spine. The surgical approach is a mini- open thoracotomy with relevance to minimally invasive deformity correction surgery for adolescent idiopathic scoliosis. To date there are no studies examining the use of this biodegradable implant in combination with biologics in a sheep thoracic spine model. METHODS. In the present study, six sheep underwent a 3-level (T6/7, T8/9 and T10/11) discectomy with randomly allocated implantation of a different graft substitute at each of the three levels; (i) calcium phosphate (CaP) coated polycaprolactone-based scaffold plus 0.54μg rhBMP-2, (ii) CaP coated PCL- based scaffold alone or (iii) autograft (mulched rib head). Fusion was assessed at six months post-surgery. RESULTS. Computed Tomographic scanning demonstrated higher fusion grades in the rhBMP-2 plus PCL- based scaffold group in comparison to either PCL-based scaffold alone or autograft. These results were supported by histological evaluations of the respective groups. Biomechanical testing revealed significantly higher stiffness for the rhBMP-2 plus PCL- based scaffold group in all loading directions in comparison to the other two groups. CONCLUSION. The results of this study demonstrate that rhBMP-2 plus PCL- based scaffold is a viable bone graft substitute, providing an optimal environment for thoracic interbody spinal fusion in a large animal model.

Optimal Bayesian experimental design for models with intractable likelihoods using indirect inference applied to biological process models

This paper addresses the problem of determining optimal designs for biological process models with intractable likelihoods, with the goal of parameter inference. The Bayesian approach is to choose a design that maximises the mean of a utility, and the utility is a function of the posterior distribution. Therefore, its estimation requires likelihood evaluations. However, many problems in experimental design involve models with intractable likelihoods, that is, likelihoods that are neither analytic nor can be computed in a reasonable amount of time. We propose a novel solution using indirect inference (II), a well established method in the literature, and the Markov chain Monte Carlo (MCMC) algorithm of Müller et al. (2004). Indirect inference employs an auxiliary model with a tractable likelihood in conjunction with the generative model, the assumed true model of interest, which has an intractable likelihood. Our approach is to estimate a map between the parameters of the generative and auxiliary models, using simulations from the generative model. An II posterior distribution is formed to expedite utility estimation. We also present a modification to the utility that allows the Müller algorithm to sample from a substantially sharpened utility surface, with little computational effort. Unlike competing methods, the II approach can handle complex design problems for models with intractable likelihoods on a continuous design space, with possible extension to many observations. The methodology is demonstrated using two stochastic models; a simple tractable death process used to validate the approach, and a motivating stochastic model for the population evolution of macroparasites.

Genetic and phenotypic evidence supports evolutionary divergence of the American flamingo (Phoenicopterus ruber) population in the Galapagos islands

The Galapagos archipelago is characterized by a high degree of endemism across many taxa, linked to the archpelago's oceanic origin and distance from other colonizing land masses. A population of ~ 500 American Flamingos (Phoenicopterus ruber) resides in Galapagos, which is thought to share an historical origin with the American Flamingo currently found in the Caribbean region. Genetic and phenotypic parameters in American Flamingos from Galapagos and from the Caribbean were investigated. Microsatellite and microchondrial DNA markers data showed that the American Flamingo population in Galapagos differs genetically from that in the Caribbean. American Flamingos in Galapagos form a clade which differs by a single common nucleotide substitution from American Flamingos in the Caribbean. The genetic differentiation is also evident from nuclear DNA in that microsatellite data reveal a number of private alleles for the American Flamingo in Galapagos. Analysis of skeletal measurements showed that American Flamingos in Galapagos are smaller than those in the Caribbean primarily due to shorter tarsus length, and differences in body shape sexual dimorphism. American Flamingo eggs from Galapagos have smaller linear dimensions and volumes than those from the Caribbean. The findings are consistent with reproductive isolation of American Flamingo population in Galapagos.