Toll-Like Receptor-2 Mediates Diet and/or Pathogen Associated Atherosclerosis: Proteomic Findings

BACKGROUND. Accumulating evidence implicates a fundamental link between the immune system and atherosclerosis. Toll-like receptors are principal sensors of the innate immune system. Here we report an assessment of the role of the TLR2 pathway in atherosclerosis associated with a high-fat diet and/or bacteria in ApoE+/- mice. METHODS AND RESULTS. To explore the role of TLR2 in inflammation- and infection-associated atherosclerosis, 10 week-old ApoE+/--TLR2+/+, ApoE+/--TLR2+/- and ApoE+/--TLR2-/- mice were fed either a high fat diet or a regular chow diet. All mice were inoculated intravenously, once per week for 24 consecutive weeks, with 50 μl live Porphyromonas gingivalis (P.g) (107 CFU) or vehicle (normal saline). Animals were euthanized 24 weeks after the first inoculation. ApoE+/--TLR2+/+ mice showed a significant increase in atheromatous lesions in proximal aorta and aortic tree compared to ApoE+/--TLR2+/- and ApoE+/--TLR2-/- mice for all diet conditions. They also displayed profound changes in plaque composition, as evidenced by increased macrophage infiltration and apoptosis, increased lipid content, and decreased smooth muscle cell mass, all reflecting an unstable plaque phenotype. SAA levels from ApoE+/--TLR2+/+ mice were significantly higher than from ApoE+/--TLR2+/- and ApoE+/--TLR2-/- mice. Serum cytokine analysis revealed increased levels of pro-inflammatory cytokines in ApoE+/--TLR2+/+ mice compared to ApoE+/--TLR2+/- and TLR2-/- mice, irrespective of diet or bacterial challenge. ApoE+/--TLR2+/+ mice injected weekly for 24 weeks with FSL-1 (a TLR2 agonist) also demonstrated significant increases in atherosclerotic lesions, SAA and serum cytokine levels compared to ApoE+/--TLR2-/- mice under same treatment condition. Finally, mass-spectrometry (MALDI-TOF-MS) of aortic samples analyzed by 2-dimentional gel electrophoresis differential display, identified 6 proteins upregulated greater than 2-fold in ApoE+/--TLR2+/+ mice fed the high fat diet and inoculated with P.g compared to any other group. CONCLUSION. Genetic deficiency of TLR2 reduces diet- and/or pathogen-associated atherosclerosis in ApoE+/- mice, along with differences in plaque composition suggesting greater structural stability while TLR-2 ligand-specific activation triggers atherosclerosis. The present data offers new insights into the pathophysiological pathways involved in atherosclerosis and paves the way for new pharmacological interventions aimed at reducing atherosclerosis.National Heart, Lung, and Blood Institute (R01 HL076801

Emerging roles for hyaluronidase in cancer metastasis and therapy

Hyaluronidases are a family of five human enzymes that have been differentially implicated in the progression of many solid tumor types, both clinically and in functional studies. Advances in the past five years have clarified many apparent contradictions, (1) by demonstrating that specific hyaluronidases have alternative substrates to hyaluronan (HA) or do not exhibit any enzymatic activity, (2) that high molecular weight HA polymers elicit signaling effects that are opposite those of the hyaluronidase-digested HA oligomers, and (3) that it is actually the combined overexpression of HA synthesizing enzymes with hyaluronidases that confers tumorigenic potential. This review examines the literature supporting these conclusions and discusses novel mechanisms by which hyaluronidases impact invasive tumor cell processes. In addition, a detailed structural and functional comparison of the hyaluronidases is presented with insights into substrate selectivity and potential for therapeutic targeting. Finally, technological advances in targeting hyaluronidase for tumor imaging and cancer therapy are summarized

Surfactant protein D inhibits HIV-1 infection of target cells via interference with gp120-CD4 interaction and modulates pro-inflammatory cytokine production

© 2014 Pandit et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Surfactant Protein SP-D, a member of the collectin family, is a pattern recognition protein, secreted by mucosal epithelial cells and has an important role in innate immunity against various pathogens. In this study, we confirm that native human SP-D and a recombinant fragment of human SP-D (rhSP-D) bind to gp120 of HIV-1 and significantly inhibit viral replication in vitro in a calcium and dose-dependent manner. We show, for the first time, that SP-D and rhSP-D act as potent inhibitors of HIV-1 entry in to target cells and block the interaction between CD4 and gp120 in a dose-dependent manner. The rhSP-D-mediated inhibition of viral replication was examined using three clinical isolates of HIV-1 and three target cells: Jurkat T cells, U937 monocytic cells and PBMCs. HIV-1 induced cytokine storm in the three target cells was significantly suppressed by rhSP-D. Phosphorylation of key kinases p38, Erk1/2 and AKT, which contribute to HIV-1 induced immune activation, was significantly reduced in vitro in the presence of rhSP-D. Notably, anti-HIV-1 activity of rhSP-D was retained in the presence of biological fluids such as cervico-vaginal lavage and seminal plasma. Our study illustrates the multi-faceted role of human SPD against HIV-1 and potential of rhSP-D for immunotherapy to inhibit viral entry and immune activation in acute HIV infection. © 2014 Pandit et al.The work (Project no. 2011-16850) was supported by Medical Innovation Fund of Indian Council of Medical Research, New Delhi, India (www.icmr.nic.in/)

WNT signalling in prostate cancer

Genome sequencing and gene expression analyses of prostate tumours have highlighted the potential importance of genetic and epigenetic changes observed in WNT signalling pathway components in prostate tumours-particularly in the development of castration-resistant prostate cancer. WNT signalling is also important in the prostate tumour microenvironment, in which WNT proteins secreted by the tumour stroma promote resistance to therapy, and in prostate cancer stem or progenitor cells, in which WNT-β-catenin signals promote self-renewal or expansion. Preclinical studies have demonstrated the potential of inhibitors that target WNT receptor complexes at the cell membrane or that block the interaction of β-catenin with lymphoid enhancer-binding factor 1 and the androgen receptor, in preventing prostate cancer progression. Some WNT signalling inhibitors are in phase I trials, but they have yet to be tested in patients with prostate cancer

Ouabain Stimulates a Na+/K+-ATPase-Mediated SFK-Activated Signalling Pathway That Regulates Tight Junction Function in the Mouse Blastocyst

The Na+/K+-ATPase plays a pivotal role during preimplantation development; it establishes a trans-epithelial ionic gradient that facilitates the formation of the fluid-filled blastocyst cavity, crucial for implantation and successful pregnancy. The Na+/K+-ATPase is also implicated in regulating tight junctions and cardiotonic steroid (CTS)-induced signal transduction via SRC. We investigated the expression of SRC family kinase (SFK) members, Src and Yes, during preimplantation development and determined whether SFK activity is required for blastocyst formation. Embryos were collected following super-ovulation of CD1 or MF1 female mice. RT-PCR was used to detect SFK mRNAs encoding Src and Yes throughout preimplantation development. SRC and YES protein were localized throughout preimplantation development. Treatment of mouse morulae with the SFK inhibitors PP2 and SU6656 for 18 hours resulted in a reversible blockade of progression to the blastocyst stage. Blastocysts treated with 10−3 M ouabain for 2 or 10 minutes and immediately immunostained for phosphorylation at SRC tyr418 displayed reduced phosphorylation while in contrast blastocysts treated with 10−4 M displayed increased tyr418 fluorescence. SFK inhibition increased and SFK activation reduced trophectoderm tight junction permeability in blastocysts. The results demonstrate that SFKs are expressed during preimplantation development and that SFK activity is required for blastocyst formation and is an important mediator of trophectoderm tight junction permeability

Reliable transfer of transcriptional gene regulatory networks between taxonomically related organisms

Baumbach J, Rahmann S, Tauch A. Reliable transfer of transcriptional gene regulatory networks between taxonomically related organisms. BMC Systems Biology. 2009;3(1):8.Background: Transcriptional regulation of gene activity is essential for any living organism. Transcription factors therefore recognize specific binding sites within the DNA to regulate the expression of particular target genes. The genome-scale reconstruction of the emerging regulatory networks is important for biotechnology and human medicine but cost-intensive, time-consuming, and impossible to perform for any species separately. By using bioinformatics methods one can partially transfer networks from well-studied model organisms to closely related species. However, the prediction quality is limited by the low level of evolutionary conservation of the transcription factor binding sites, even within organisms of the same genus. Results: Here we present an integrated bioinformatics workflow that assures the reliability of transferred gene regulatory networks. Our approach combines three methods that can be applied on a large-scale: re-assessment of annotated binding sites, subsequent binding site prediction, and homology detection. A gene regulatory interaction is considered to be conserved if (1) the transcription factor, (2) the adjusted binding site, and (3) the target gene are conserved. The power of the approach is demonstrated by transferring gene regulations from the model organism Corynebacterium glutamicum to the human pathogens C. diphtheriae, C. jeikeium, and the biotechnologically relevant C. efficiens. For these three organisms we identified reliable transcriptional regulations for similar to 40% of the common transcription factors, compared to similar to 5% for which knowledge was available before. Conclusion: Our results suggest that trustworthy genome-scale transfer of gene regulatory networks between organisms is feasible in general but still limited by the level of evolutionary conservation

Enzyme immobilisation on amino-functionalised multi-walled carbon nanotubes : structural and biocatalytic characterisation

BACKGROUND: The aim of this work is to investigate the structure and function of enzymes immobilised on nanomaterials. This work will allow better understanding of enzyme-nanomaterial interactions, as well as designing functional protein-nanomaterial conjugates. METHODOLOGY/PRINCIPAL FINDINGS: Multiwalled carbon nanotubes (MWNTs) were functionalised with amino groups to improve solubility and biocompatibility. The pristine and functionalised forms of MWNTs were characterised with Fourier-transform infrared spectroscopy. Thermogravimetric analysis was done to examine the degree of the functionalisation process. An immobilised biocatalyst was prepared on functionalised nanomaterial by covalent binding. Thermomyces lanuginosus lipase was used as a model enzyme. The structural change of the immobilised and free lipases were characterised with transmission electron Microscopy, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy and Circular dichroism spectroscopy. Biochemical characterisation of immobilised enzyme showed broader pH and thermal optima compared to soluble form. Reusability of the immobilised enzyme for hydrolysis of long chain esters was demonstrated up to ten cycles. CONCLUSION/SIGNIFICANCE: Lipase immobilised on MWNTs has exhibited significantly improved thermal stability. The exploration of advanced nanomaterial for enzyme immobilisation support using sophisticated techniques makes nanobiocatalyst of potential interest for biosensor applications

Indeterminacy of Reverse Engineering of Gene Regulatory Networks: The Curse of Gene Elasticity

Gene Regulatory Networks (GRNs) have become a major focus of interest in recent years. A number of reverse engineering approaches have been developed to help uncover the regulatory networks giving rise to the observed gene expression profiles. However, this is an overspecified problem due to the fact that more than one genotype (network wiring) can give rise to the same phenotype. We refer to this phenomenon as “gene elasticity.” In this work, we study the effect of this particular problem on the pure, data-driven inference of gene regulatory networks.We simulated a four-gene network in order to produce “data” (protein levels) that we use in lieu of real experimental data. We then optimized the network connections between the four genes with a view to obtain the original network that gave rise to the data. We did this for two different cases: one in which only the network connections were optimized and the other in which both the network connections as well as the kinetic parameters (given as reaction probabilities in our case) were estimated. We observed that multiple genotypes gave rise to very similar protein levels. Statistical experimentation indicates that it is impossible to differentiate between the different networks on the basis of both equilibrium as well as dynamic data.We show explicitly that reverse engineering of GRNs from pure expression data is an indeterminate problem. Our results suggest the unsuitability of an inferential, purely data-driven approach for the reverse engineering transcriptional networks in the case of gene regulatory networks displaying a certain level of complexity