Dissection of a metastatic gene expression signature into distinct components

Background

Metastasis, the process whereby cancer cells spread, is in part caused by an incompletely understood interplay between cancer cells and the surrounding stroma. Gene expression studies typically analyze samples containing tumor cells and stroma. Samples with less than 50% tumor cells are generally excluded, thereby reducing the number of patients that can benefit from clinically relevant signatures.

Results

For a head-neck squamous cell carcinoma (HNSCC) primary tumor expression signature that predicts the presence of lymph node metastasis, we first show that reduced proportions of tumor cells results in decreased predictive accuracy. To determine the influence of stroma on the predictive signature and to investigate the interaction between tumor cells and the surrounding microenvironment, we used laser capture microdissection to divide the metastatic signature into six distinct components based on tumor versus stroma expression and on association with the metastatic phenotype. A strikingly skewed distribution of metastasis associated genes is revealed.

Conclusion

Dissection of predictive signatures into different components has implications for design of expression signatures and for our understanding of the metastatic process. Compared to primary tumors that have not formed metastases, primary HNSCC tumors that have metastasized are characterized by predominant down-regulation of tumor cell specific genes and exclusive up-regulation of stromal cell specific genes. The skewed distribution agrees with poor signature performance on samples that contain less than 50% tumor cells. Methods for reducing tumor composition bias that lead to greater predictive accuracy and an increase in the types of samples that can be included are presented.     

Regulation of SMC traction forces in human aortic thoracic aneurysms

Smooth muscle cells (SMCs) usually express a contractile phenotype in the healthy aorta. However, aortic SMCs have the ability to undergo profound changes in phenotype in response to changes in their extracellular environment, as occurs in ascending thoracic aortic aneurysms (ATAA). Accordingly, there is a pressing need to quantify the mechanobiological effects of these changes at single cell level. To address this need, we applied Traction Force Microscopy (TFM) on 759 cells coming from three primary healthy (AoPrim) human SMC lineages and three primary aneurysmal (AnevPrim) human SMC lineages, from age and gender matched donors. We measured the basal traction forces applied by each of these cells onto compliant hydrogels of different stiffness (4, 8, 12, 25 kPa). Although the range of force generation by SMCs suggested some heterogeneity, we observed that: 1. the traction forces were significantly larger on substrates of larger stiffness; 2. traction forces in AnevPrim were significantly higher than in AoPrim cells. We modelled computationally the dynamic force generation process in SMCs using the motor-clutch model and found that it accounts well for the stiffness-dependent traction forces. The existence of larger traction forces in the AnevPrim SMCs were related to the larger size of cells in these lineages. We conclude that phenotype changes occurring in ATAA, which were previously known to reduce the expression of elongated and contractile SMCs (rendering SMCs less responsive to vasoactive agents), tend also to induce stronger SMCs. Future work aims at understanding the causes of this alteration process in aortic aneurysms.

     

Phosphorus acquisition from phytate depends on efficient bacterial grazing, irrespective of the mycorrhizal status of Pinus pinaster

Background and aims

Phosphorus from phytate, although constituting the main proportion of organic soil P, is unavailable to plants. Despite the well-known effects of rhizosphere trophic relationships on N mineralization, no work has been done yet on P mineralization. We hypothesized that the interactions between phytate-mineralizing bacteria, mycorrhizal fungi and bacterial grazer nematodes are able to improve plant P use from phytate.

Methods

We tested this hypothesis by growing Pinus pinaster seedlings in agar containing phytate as P source. The plants, whether or not ectomycorrhizal with the basidiomycete Hebeloma cylindrosporum, were grown alone or with a phytase-producing bacteria Bacillus subtilis and two bacterial-feeder nematodes, Rhabditis sp. and Acrobeloides sp. The bacteria and the nematodes were isolated from ectomycorrhizal roots and soil from P. pinaster plantations.

Results

Only the grazing of bacteria by nematodes enhanced plant P accumulation. Although plants increased the density of phytase-producing bacteria, these bacteria alone did not improve plant P nutrition. The seedlings, whether ectomycorrhizal or not, displayed a low capacity to use P from phytate.

Conclusions

In this experiment, the bacteria locked up the phosphorus, which was delivered to plant only by bacterial grazers like nematodes. Our results open an alternative route for better utilization of poorly available organic P by plants.     

Combined Approaches for Drug Design Points the Way to Novel Proline Racemase Inhibitor Candidates to Fight Chagas’ Disease

Chagas’ disease is caused by Trypanosoma cruzi, a protozoan transmitted to humans by blood-feeding insects, blood transfusion or congenitally. Previous research led us to discover a parasite proline racemase (TcPRAC) and to establish its validity as a target for the design of new chemotherapies against the disease, including its chronic form. A known inhibitor of proline racemases, 2-pyrrolecarboxylic acid (PYC), is water-insoluble. We synthesized soluble pyrazole derivatives, but they proved weak or inactive TcPRAC inhibitors. TcPRAC catalytic site is too small and constrained when bound to PYC to allow efficient search for new inhibitors by virtual screening. Forty-nine intermediate conformations between the opened enzyme structure and the closed liganded one were built by calculating a transition path with a method we developed. A wider range of chemical compounds could dock in the partially opened intermediate active site models in silico. Four models were selected for known substrates and weak inhibitors could dock in them and were used to screen chemical libraries. Two identified soluble compounds, (E)-4-oxopent-2-enoic acid (OxoPA) and its derivative (E)-5-bromo-4-oxopent-2-enoic acid (Br-OxoPA), are irreversible competitive inhibitors that presented stronger activity than PYC on TcPRAC. We show here that increasing doses of OxoPA and Br-OxoPA hamper T. cruzi intracellular differentiation and fate in mammalian host cells. Our data confirm that through to their binding mode, these molecules are interesting and promising as lead compounds for the development of chemotherapies against diseases where active proline racemases play essential roles.     

Treatment with the arginase inhibitor Nw-hydroxy-nor-L-arginine restores endothelial function in rat adjuvant-induced arthritis

Introduction

Endothelial dysfunction (ED) participates to atherogenesis associated to rheumatoid arthritis. We recently reported increased arginase activity/expression in vessels from adjuvant-induced arthritis (AIA) rats. In the present study, we investigated the effects of a curative treatment with the arginase inhibitor N_w-hydroxy-nor-L-arginine (nor-NOHA) on vascular dysfunction in AIA rats.

Methods

AIA rats were treated with nor-NOHA (40 mg/kg/d, ip) for 21 days after the onset of arthritis. A group of untreated AIA rats and a group of healthy rats served as controls. ED was assessed by the vasodilatory effect of acetylcholine (Ach) on aortic rings. The role of superoxide anions, prostanoids, endothelium-derived hyperpolarizing factor (EDHF) and nitric oxide synthase (NOS) pathway was studied. Plasma levels of IL-6 and vascular endothelial growth factor (VEGF) were determined by ELISA kits. Arthritis severity was estimated by a clinical, radiological and histological analysis.

Results

Nor-NOHA treatment fully restored the aortic response to Ach to that of healthy controls. The results showed that this beneficial effect is mediated by an increase in NOS activity and EDHF and reduced superoxide anion production as well as a decrease in the activity of cyclooxygenase (COX)-2, thromboxane and prostacyclins synthases. In addition, nor-NOHA decreased IL-6 and VEGF plasma levels in AIA rats. By contrast, the treatment did not modify arthritis severity in AIA rats.

Conclusions

The treatment with an arginase inhibitor has a potent effect on ED in AIA independently of the severity of the disease. Our results suggest that this new pharmacological approach has the potential as a novel add-on therapy in the treatment of RA.     

Plant–Environment Interactions in the Low Arctic Torngat Mountains of Labrador

Ecosystems - The eastern Canadian Subarctic and Arctic are experiencing significant environmental change with widespread implications for the people, plants, and animals living there. In this...     

Proteomic studies highlight outer‐membrane proteins related to biofilm development in the marine bacterium Pseudoalteromonas sp. D41

Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer‐membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer‐membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB‐dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT‐PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment.     

Isolation and physiological study of an amylolytic strain of Lactobacillus plantarum

Summary An amylolytic lactic acid bacterium identified as Lactobacillus plantarum was isolated from cassava roots (Manihot esculenta var. Ngansa) during reting. The amylolytic enzyme synthesized was an extracellular -amylase with an optimum pH of 5.0 and an optimum temperature of 55° C. Cultured on starch, the strain displayed a growth rate of 0.43 h^–1, a biomass yield of 0.19 g·g^–1 and a lactate yield of 0.81 g·g^–1. The growth kinetics were similar on starch and glucose. Sufficient enzyme was synthesized and starch hydrolysis was not a limiting factor for growth. Biosynthesis of the enzyme was observed when the glucose concentration was less than 6.7 g·l^–1 and reached up to 4 IU·ml^–1 at the end of the fermentation. Offprint requests to: M. Raimbault     

Distribution of NO3− reduction between roots and shoots of peachtree seedlings as affected by NO3− uptake rate

The distribution of NO₃^? reduction between roots and shoots was studied in hydro-ponically-grown peach-tree seedlings (Prunus persica L.) during recovery from N starvation. Uptake, translocation and reduction of NO₃^?, together with transport through xylem and phloem of the newly reduced N were estimated, using ¹⁵N labellings, in intact plants supplied for 90 h with 0.5 mM NH₄⁺ and 0.5, 1.5 or 10 mM NO₃^?. Xylem transport of NO₃^? was further investigated by xylem sap analysis in a similar experiment. The roots were the main site of NO₃^? reduction at all 3 levels of NO₃^? nutrition. However, the contribution of the shoots to the whole plant NO₃^? reduction increased with increasing external NO₃^? availability. This contribution was estimated to be 20, 23 and 42% of the total assimilation at 0.5, 1.5 and 10 mM NO₃^?, respectively. Both ¹⁵N results and xylem sap analysis confirmed that this trend was due to an enhancement of NO₃^? translocation from roots to shoots. It is proposed that the lack of NO₃^? export to the shoots at low NO₃^? uptake rate resulted from a competition between NO₃^? reduction in the root epidermis/cortex and NO₃^? diffusion to the stele. On the other hand, net xylem transport of newly reduced N was very efficient since ca 70% of the amino acids synthesized in the roots were translocated to the shoots, regardless of the level of NO₃^? nutrition. This net xylem transport by far exceeded the net downward phloem transport of the reduced N assimilated in shoots. As a consequence, the reduced N resulting from NO₃^? assimilation, principally occurring in the roots, was mainly incorporated in the shoots.     

The metabolic blueprint of Phaeodactylum tricornutum reveals a eukaryotic Entner-Doudoroff glycolytic pathway

Diatoms are one of the most successful groups of unicellular eukaryotic algae. Successive endosymbiotic events contributed to their flexible metabolism, making them competitive in variable aquatic habitats. Although the recently sequenced genomes of the model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana have provided the first insights into their metabolic organization, the current knowledge on diatom biochemistry remains fragmentary. By means of a genome‐wide approach, we developed DiatomCyc, a detailed pathway/genome database of P. tricornutum. DiatomCyc contains 286 pathways with 1719 metabolic reactions and 1613 assigned enzymes, spanning both the central and parts of the secondary metabolism of P. tricornutum. Central metabolic pathways, such as those of carbohydrates, amino acids and fatty acids, were covered. Furthermore, our understanding of the carbohydrate model in P. tricornutum was extended. In particular we highlight the discovery of a functional Entner–Doudoroff pathway, an ancient alternative for the glycolytic Embden–Meyerhof–Parnas pathway, and a putative phosphoketolase pathway, both uncommon in eukaryotes. DiatomCyc is accessible online ( http://www.diatomcyc.org ), and offers a range of software tools for the visualization and analysis of metabolic networks and ‘omics’ data. We anticipate that DiatomCyc will be key to gaining further understanding of diatom metabolism and, ultimately, will feed metabolic engineering strategies for the industrial valorization of diatoms.