Cellular automaton simulation examining progenitor hierarchy structure effects on mammary ductal carcinoma in situ

A computer simulation is used to model ductal carcinoma in situ, a form of non-invasive breast cancer. The simulation uses known histological morphology, cell types, and stochastic cell proliferation to evolve tumorous growth within a duct. The ductal simulation is based on a hybrid cellular automaton design using genetic rules to determine each cell's behavior. The genetic rules are a mutable abstraction that demonstrate genetic heterogeneity in a population. Our goal was to examine the role (if any) that recently discovered mammary stem cell hierarchies play in genetic heterogeneity, DCIS initiation and aggressiveness. Results show that simpler progenitor hierarchies result in greater genetic heterogeneity and evolve DCIS significantly faster. However, the more complex progenitor hierarchy structure was able to sustain the rapid reproduction of a cancer cell population for longer periods of time.     

First step of the cell-penetrating peptide mechanism involves Rac1 GTPase-dependent actin-network remodelling

BACKGROUND INFORMATION: Application of CPPs (cell-penetrating peptides) constitutes a promising strategy for the intracellular delivery of therapeutic molecules. The non-covalent approach based on the amphipathic peptide MPG has been successfully used to improve the delivery of biologically active macromolecules, both in cellulo and in vivo, through a mechanism independent of the endosomal pathway and mediated by the membrane potential. RESULTS: In the present study, we have investigated the first step of the cellular uptake mechanism of MPG and shown that both MPG and MPG-cargo complexes interact with the extracellular matrix through the negatively charged heparan sulfate proteoglycans. We demonstrated that initiation of cellular uptake constitutes a highly dynamic mechanism where the binding of MPG or the MPG-cargo to the extracellular matrix is rapidly followed by a remodelling of the actin network associated with the activation of the GTPase Rac1. We suggest that MPG-induced clustering of the glycosaminoglycan platform constitutes the 'onset' of the cellular uptake mechanism, thereby increasing membrane dynamics and membrane fusion processes. This process favours cell entry of MPG or MPG-DNA complexes, which is further controlled by the ability of MPG to induce a local membrane destabilization. CONCLUSIONS: Although CPPs are taken up through different pathways and mechanisms, the initial step involves electrostatic interactions with the glycosaminoglycan platform, and the dynamics of associated membrane microdomains can be generalized to most non-viral delivery systems.     

Understanding contributions of cohort effects to growth rates of fluctuating populations

1. Understanding contributions of cohort effects to variation in population growth of fluctuating populations is of great interest in evolutionary biology and may be critical in contributing towards wildlife and conservation management. Cohort-specific contributions to population growth can be evaluated using age-specific matrix models and associated elasticity analyses. 2. We developed age-specific matrix models for naturally fluctuating populations of stoats Mustela erminea in New Zealand beech forests. Dynamics and productivity of stoat populations in this environment are related to the 3-5 year masting cycle of beech trees and consequent effects on the abundance of rodents. 3. The finite rate of increase (lambda) of stoat populations in New Zealand beech forests varied substantially, from 1.98 during seedfall years to 0.58 during post-seedfall years. Predicted mean growth rates for stoat populations in continuous 3-, 4- or 5-year cycles are 0.85, 1.00 and 1.13. The variation in population growth was a consequence of high reproductive success of females during seedfall years combined with low survival and fertility of females of the post-seedfall cohort. 4. Variation in population growth was consistently more sensitive to changes in survival rates both when each matrix was evaluated in isolation and when matrices were linked into cycles. Relative contributions to variation in population growth from survival and fertility, especially in 0-1-year-old stoats, also depend on the year of the cycle and the number of transitional years before a new cycle is initiated. 5. Consequently, management strategies aimed at reducing stoat populations that may be best during one phase of the beech seedfall cycle may not be the most efficient during other phases of the cycle. We suggest that management strategies based on elasticities of vital rates need to consider how population growth rates vary so as to meet appropriate economic and conservation targets.     

Colocalization of ferroportin-1 with hephaestin on the basolateral membrane of human intestinal absorptive cells

An iron exporter ferroportin-1 (FPN-1) and a multi-copper oxidase hephaestin (Heph) are predicted to be expressed on the basolateral membrane of the enterocyte and involved in the processes of iron export across the basolateral membrane of the enterocyte. However, it is not clear where these proteins are exactly located in the intestinal absorptive cell. We examined cellular localization of FPN-1 and Heph in the intestinal absorptive cells using the fully differentiated Caco-2 cells. Confocal microscope study showed that FPN-1 and Heph are located on the basolateral membrane and they are associated with the transferrin receptor (TfR) in fully differentiated Caco-2 cells grown on microporous membrane inserts. However, Heph protein was not detected in the crypt cell-like proliferating Caco-2 cell. In stably transfected human intestinal absorptive cells expressing human FPN-1 modified by the addition of GFP at the C-terminus, we show that FPN-1-GFP is located on the basolateral membrane and it is associated with Heph suggesting the possibility that FPN-1 might associate and interact with Heph in the process of iron exit across the basolateral membrane of intestinal absorptive cell.     

The Porphyromonas gingivalis clpB gene is involved in cellular invasion in vitro and virulence in vivo

ClpB, a component of stress response in microorganisms, serves as a chaperone, preventing protein aggregation and assisting in the refolding of denatured proteins. A clpB mutant of Porphyromonas gingivalis W83 demonstrated increased sensitivity to heat stress, but not to hydrogen peroxide and extreme pHs. In KB cells, human coronary artery endothelial (HCAE) cells and gingival epithelial cells, the clpB mutant exhibited significantly decreased invasion suggesting that the ClpB protein is involved in cellular invasion. Transmission electron microscopic analysis showed that the clpB mutant was more susceptible to intracellular killing than the wild-type strain in HCAE cells. The global genetic profile of the clpB mutant showed that 136 genes belonging to several different cellular function groups were differentially regulated, suggesting that ClpB is ultimately involved in the expression of multiple P. gingivalis genes. A competition assay in which a mixture of wild-type W83 and the clpB mutant were injected into mice demonstrated that the clpB mutant did not survive as well as the wild type. Additionally, mice treated with the clpB mutant alone survived significantly better than those treated with the wild-type strain. Collectively, these data suggest that ClpB, either directly or indirectly, plays an important role in P. gingivalis virulence.     

Adjuvant effect of CIA07, a combination of Escherichia coli DNA fragments and modified lipopolysaccharides, on the immune response to hepatitis B virus surface antigen

CIA07 is an immunostimulatory agent composed of bacterial DNA fragments and modified lipopolysaccharide, which has antitumor activity against bladder cancer in mice. In this study, the adjuvant activity of CIA07 was evaluated using hepatitis B virus surface antigen (HBsAg) as the immunogen. Mice were immunized intramuscularly three times at 1-week intervals with HBsAg alone or in combination with alum, bacterial DNA fragments, modified lipopolysaccharide, CIA07 or CpG1826, and immune responses were assessed. At 1 week after the final injection, the HBsAg-specific total serum IgG antibody titer in CIA07-treated mice was 14 times higher than that in animals administered antigen alone, six times higher than in mice given alum or bacterial DNA fragments and twice as high as those treated with modified lipopolysaccharide or CpG1826, and remained maximal until 8 weeks postimmunization. Animals receiving antigen alone or plus alum displayed barely detectable HBsAg-specific serum IgG2a antibody responses. However, coadministration of CIA07 with antigen led to markedly enhanced serum IgG2a antibody titer and IFN-gamma(+) production in splenocytes, indicating that CIA07 effectively induces Th1-type immune responses. In addition, the number of HBsAg-specific CD8(+) T cells in peripheral blood mononuclear cells was elevated in CIA07-treated mice. These data clearly demonstrate that CIA07 is able to induce both cellular and humoral immune responses to HBsAg, and confirm its potential as an adjuvant in therapeutic vaccines for hepatitis B virus infections.     

Increased polyploidy in aortic vascular smooth muscle cells during aging is marked by cellular senescence

We previously reported that the frequency of polyploid aortic vascular smooth muscle cells (VSMC) serves as a biomarker of aging. Cellular senescence of somatic cells is another marker of aging that is characterized by the inability to undergo cell division. Here, we examined whether polyploidy is associated with the development of cellular senescence in vivo. Analysis of aortic tissue preparations from young and old Brown Norway rats showed that expression of senescence markers such as p16(INK4a) and senescence-associated beta-galactosidase activity are detected primarily in the old tissues. VSMC from p16(INK4a) knockout and control mice display similar levels of polyploid cells. Intriguingly, senescence markers are expressed in most, but not all, polyploid VSMC. Moreover, the polyploid cells exhibit limited proliferative capacity in comparison to their diploid counterparts. This study is the first to demonstrate in vivo that polyploid VSMC adopt a senescent phenotype.     

Mir-190b negatively contributes to the Trypanosoma cruzi- infected cell survival by repressing PTEN protein expression

Chagas disease, which is caused by the intracellular protozoanTrypanosoma cruzi, is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol-3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells.     

Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs)

The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly.     

Integrins Influence the Size and Dynamics of Signaling Microclusters in a Pyk2-dependent Manner

Integrin engagement on lymphocytes initiates “outside-in” signaling that is required for cytoskeleton remodeling and the formation of the synaptic interface. However, the mechanism by which the “outside-in” signal contributes to receptor-mediated intracellular signaling that regulates the kinetics of granule delivery and efficiency of cytolytic activity is not well understood. We have found that variations in ICAM-1 expression on tumor cells influence killing kinetics of these cells by CD16.NK-92 cytolytic effectors suggesting that changes in integrin ligation on the effector cells regulate the kinetics of cytolytic activity by the effector cells. To understand how variations of the integrin receptor ligation may alter cytolytic activity of CD16.NK-92 cells, we analyzed molecular events at the contact area of these cells exposed to planar lipid bilayers that display integrin ligands at different densities and activating CD16-specific antibodies. Changes in the extent of integrin ligation on CD16.NK-92 cells at the cell/bilayer interface revealed that the integrin signal influences the size and the dynamics of activating receptor microclusters in a Pyk2-dependent manner. Integrin-mediated changes of the intracellular signaling significantly affected the kinetics of degranulation of CD16.NK-92 cells providing evidence that integrins regulate the rate of target cell destruction in antibody-dependent cell cytotoxicity (ADCC).