Caveolin-1 overexpression correlates with tumour progression markers in pancreatic ductal adenocarcinoma

The assessment of caveolin-1 (Cav-1) as a marker of tumor aggressiveness in pancreatic ductal adenocarcinoma (PDAC). In this study, we examined the expression of Cav-1 in 34 human PDAC tissue samples and the associated peritumoral tissues by immunohistochemistry and western blot. Additionally, we correlated Cav-1 expression with other tissue (Ki-67, p53) and serum (CA 19-9) tumor markers. In the tumor-derived tissue, both tumor cells and blood vessels expressed Cav-1. In contrast, in peritumoral tissue, Cav-1 expression was confined mainly to blood vessels and was only occasionally expressed in ductal or parenchymal cells. Western blot analysis confirmed the overexpression of Cav-1 in pancreatic tumors compared with peritumoral tissue. Cav-1 expression in tumor tissues was correlated with both the Ki-67 LI (r = 0.95, P < 0.0001) and p53 expression (χ2 = 9.91, P < 0.005). Overexpression of Cav-1 was associated with tumor size, grade and stage and Cav-1 expression in tumors was correlated with an increased serum level of CA 19-9 (r = 0.795, P < 0.001). Based on the results of this study, the inclusion of Cav-1 in a putative panel of biomarkers predicting pancreatic cancer aggressiveness is warranted.     

Antibody Targeting of Cathepsin S Inhibits Angiogenesis and Synergistically Enhances Anti-VEGF

Background

Angiogenesis is a key hallmark of tumourigenesis and its inhibition is a proven strategy for the development of novel anti-cancer therapeutics. An important aspect of early angiogenesis is the co-ordinated migration and invasion of endothelial cells through the hypoxic tumour tissue. Cathepsin S has been shown to play an important role in angiogenesis as has vascular endothelial growth factor (VEGF). We sought to assess the anti-angiogenic effect of Fsn0503, a novel cathepsin S inhibitory antibody, when combined with anti-VEGF on vascular development.

Methodology/Principal Findings

Cathepsin S expression and secretion from endothelial cells was characterised using RT-PCR and western blotting. We further show that cathepsin S promotes pericellular hydrolysis of extracellular matrix components in the tumour microenvironment and facilitates endothelial invasion. The cathepsin S inhibitory antibody, Fsn0503, blocks extracellular proteolysis, inhibiting endothelial invasion and tube formation in cell-based assays. The anti-angiogenic effects of Fsn0503 were also shown in vivo where it significantly retarded the development of vasculature in human xenograft models. Furthermore, when Fsn0503 was combined with an anti-VEGF antibody, a synergistic inhibition of microvascular development was observed.

Conclusions/Significance

Taken together, this data demonstrates that the antibody-mediated targeting of cathepsin S represents a novel method of inhibiting angiogenesis. Furthermore, when used in combination with anti-VEGF therapies, Fsn0503 has the potential to significantly enhance current treatments of tumour neovascularisation and may also be of use in the treatment of other conditions associated with inappropriate angiogenesis.     

Oligomeric structure of ExbB and ExbB-ExbD isolated from Escherichia coli as revealed by LILBID mass spectrometry

Energy-coupled transporters in the outer membrane of Escherichia coli and other Gram-negative bacteria allow the entry of scarce substrates, toxic proteins, and bacterial viruses (phages) into the cells. The required energy is derived from the proton-motive force of the cytoplasmic membrane, which is coupled to the outer membrane via the ExbB-ExbD-TonB protein complex. Knowledge of the structure of this complex is required to elucidate the mechanisms of energy harvesting in the cytoplasmic membrane and energy transfer to the outer membrane transporters. Here we solubilized an ExbB oligomer and an ExbB-ExbD subcomplex from the cytoplasmic membrane with the detergent undecyl maltoside. Using laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS), we determined at moderate desorption laser energies the oligomeric structure of ExbB to be mainly hexameric (ExbB(6)), with minor amounts of trimeric (ExbB(3)), dimeric (ExbB(2)), and monomeric (ExbB(1)) oligomers. Under the same conditions ExbB-ExbD formed a subcomplex consisting of ExbB(6)ExbD(1), with a minor amount of ExbB(5)ExbD(1). At higher desorption laser intensities, ExbB(1) and ExbD(1) and traces of ExbB(3)ExbD(1), ExbB(2)ExbD(1), ExbB(1)ExbD(1), ExbB(3), and ExbB(2) were observed. Since the ExbB(6) complex and the ExbB(6)ExbD(1) complex remained stable during solubilization and subsequent chromatographic purification on nickel-nitrilotriacetate agarose, Strep-Tactin, and Superdex 200, and during native blue gel electrophoresis, we concluded that ExbB(6) and ExbB(6)ExbD(1) are subcomplexes on which the final complex including TonB is assembled.     

Optical method for monitoring of photodynamic inactivation of bacteria

Photodynamic inactivation is a new promising approach to treat bacterial infections. Usually, the evaluation of the efficacy of this method is done through time-consuming and labor-intensive microbiological test methods. This paper describes the development and implementation of an optical method to evaluate the photodynamic inactivation of bacteria based on non-invasive diffuse reflectance measurements. Five Staphylococcus aureus cultures and 15 mice have been used in this study. A skin lesion was created on the back of all animals, and it was contaminated with S. aureus (5.16 ± 0.013 log CFU/ml). Toluidine Blue O (c = 8.67 × 10 ^− 3 M) has been used as a photosensitiser agent. The bacterial cultures and animals were exposed to laser radiation (λ = 635 nm, P = 15 mW, DE = 8.654 J/cm²) for 20 min. The photodynamic inactivation of bacteria was monitored by acquiring the wounds’ reflection spectra at different time points and by microbiological exams on the bioptical material. The good correlation between the diffuse reflectance and colony-forming units demonstrates the value of this optical method based on diffuse reflectance measurements as a rapid technique to monitor photodynamic bacterial inactivation.     

IFN-α mediates the development of autoimmunity both by direct tissue toxicity and through immune cell recruitment mechanisms

IFN-α is known to play a key role in autoimmunity, but the mechanisms are uncertain. Although the induction of autoimmunity by IFN-α is consistent with primarily immunomodulatory effects, the high frequency of nonautoimmune inflammation suggests other mechanisms. We used thyroiditis as a model to dissect these possibilities. IFN-α treatment of cultured thyrocytes increased expression of thyroid differentiation markers, thyroglobulin, thyroid-stimulating hormone receptor, thyroid peroxidase, and sodium iodide transporter. RNAseq analysis demonstrated that pathways of Ag presentation, pattern recognition receptors, and cytokines/chemokines were also stimulated. These changes were associated with markedly increased nonapoptotic thyroid cell death, suggesting direct toxicity. To corroborate these in vitro findings, we created transgenic mice with thyroid-specific overexpression of IFN-α under control of the thyroglobulin promoter. Transgenic mice developed marked inflammatory thyroid destruction associated with immune cell infiltration of thyroid and surrounding tissues leading to profound hypothyroidism, findings consistent with our in vitro results. In addition, transgenic mice thyroids showed upregulation of pathways similar to those observed in cultured thyrocytes. In particular, expression of granzyme B, CXCL10, a subset of the tripartite motif-containing family, and other genes involved in recruitment of bystander cytotoxic immune responses were increased. Pathways associated with apoptosis and autophagy were not induced. Taken together, our data demonstrate that the induction of tissue inflammation and autoimmunity by IFN-α involves direct tissue toxic effects as well as provocation of destructive bystander immune responses.     

Structure of a xanthine oxidase-related 4-hydroxybenzoyl-CoA reductase with an additional [4Fe-4S] cluster and an inverted electron flow

The Mo-flavo-Fe/S-dependent heterohexameric protein complex 4-hydroxybenzoyl-CoA reductase (4-HBCR, dehydroxylating) is a central enzyme of the anaerobic degradation of phenolic compounds and belongs to the xanthine oxidase (XO) family of molybdenum enzymes. Its X-ray structure was established at 1.6 A resolution. The most pronounced difference between 4-HBCR and other structurally characterized members of the XO family is the insertion of 40 amino acids within the beta subunit, which carries an additional [4Fe-4S] cluster at a distance of 16.5 A to the isoalloxazine ring of FAD. The architecture of 4-HBCR and concomitantly performed electron transfer rate calculations suggest an inverted electron transfer chain from the donor ferredoxin via the [4Fe-4S] cluster to the Mo over a distance of 55 A. The binding site of 4-hydroxybenzoyl-CoA is located in an 18 A long channel lined up by several aromatic side chains around the aromatic moiety, which are proposed to shield and stabilize the postulated radical intermediates during catalysis.     

A cradle-to-gate life cycle assessment of wood fibre-reinforced polylactic acid (PLA) and polylactic acid/thermoplastic starch (PLA/TPS) biocomposites

Purpose

Biopolymers are considered to be environmentally friendlier than petroleum-based polymers, but little is known about their environmental performance against petroleum-based products. This paper presents the results of a life cycle assessment (LCA) of two prototype biocomposite formulations produced by extrusion of wood fibre with either polylactic acid (PLA) or a blend of PLA and locally produced thermoplastic starch (TPS).

Methods

The study followed the LCA methodology outlined in the two standards set out by the International Organization for Standardization (ISO): ISO 14040 and ISO 14044 of 2006. A life cycle inventory (LCI) for the biocomposite formulations was developed, and a contribution analysis was performed to identify the significant inputs. Environmental performances of the two formulations were then compared with each other and polypropylene (PP), a petroleum-based polymer. The US Environmental Protection Agency’s impact assessment method, “TRACI: The Tool for the Reduction and Assessment of Chemical and Other Environmental Impacts”, was combined with Cumulative Energy Demand (a European method) in order to characterize the inventory flows. Environmental impact categories chosen for the analysis were the following: global warming, stratospheric ozone depletion, acidification of land and water, eutrophication, smog, human health (respiratory, carcinogenic, and non-carcinogenic) effects and ecotoxicity.

Results and discussion

We found that PLA is the significant input which contributes mostly to fossil fuel consumption, acidification and respiratory and smog effects. Impacts from PLA transport from the faraway source significantly added more burden to its contributions. TPS causes less environmental burden compared to PLA; the environmental performance of the biocomposite improved when a blend of PLA and TPS is used in formulating the biocomposite. The two formulations performed better than PP in all the environmental impact categories except eutrophication effects, which is important on a regional basis.

Conclusions

The following conclusions were drawn from this study:

• PLA is the environmentally significant input among the three raw materials.
• TPS causes less environmental burden than PLA. Environmental performance of the biocomposite improves in the life cycle energy consumption, fossil energy use, ozone depletion and non-carcinogenic impact categories when a blend of PLA and TPS is used.
• The biocomposite can outperform PP in all the impact categories except eutrophication effects if manufactured using hydroelectricity.

The biopolymer could be a potential alternative to PP as it could cause less of a burden to the environment on a cradle-to-gate basis. Environmental impacts at the complete life cycle levels should be looked into in order to fully understand its potential.     

Structural and functional implications of the QUA2 domain on RNA recognition by GLD-1

The STAR family comprises ribonucleic acid (RNA)-binding proteins that play key roles in RNA-regulatory processes. RNA recognition is achieved by a KH domain with an additional α-helix (QUA2) that seems to extend the RNA-binding surface to six nucleotides for SF1 (Homo sapiens) and seven nucleotides for GLD-1 (Caenorhabditis elegans). To understand the structural basis of this probable difference in specificity, we determined the solution structure of GLD-1 KH-QUA2 with the complete consensus sequence identified in the tra-2 gene. Compared to SF1, the GLD-1 KH-QUA2 interface adopts a different conformation resulting indeed in an additional sequence-specific binding pocket for a uracil at the 5′end. The functional relevance of this binding pocket is emphasized by our bioinformatics analysis showing that GLD-1 binding sites with this 5′end uracil are more predictive for the functional response of the messenger RNAs to gld-1 knockout. We further reveal the importance of the KH-QUA2 interface in vitro and that its alteration in vivo affects the level of translational repression dependent on the sequence of the GLD-1 binding motif. In conclusion, we demonstrate that the QUA2 domain distinguishes GLD-1 from other members of the STAR family and contributes more generally to the modulation of RNA-binding affinity and specificity of KH domain containing proteins.