Mapping functional associations in the entire genome of Drosophila melanogaster using fusion analysis

We have previously shown that the detection of gene fusion events can contribute towards the elucidation of functional associations of proteins within entire genomes. Here we have analysed the entire genome of Drosophila melanogaster using fusion analysis and two additional constraints that improve the reliability of the predictions, viz. low sequence similarity and low degree of paralogy of the component proteins involved in a fusion event. Imposing these constraints, the total number of unique component pairs is reduced from 18 654 to a mere 220 cases, which are expected to represent some of the most reliably detected functionally associated proteins. Using additional information from sequence databases, we have been able to detect pairs of functionally associated proteins with important functions in cellular and developmental pathways, such as spermatogenesis and programmed cell death.     

Thyroid hormone and cardioprotection: Study of p38 MAPK and JNKs during ischaemia and at reperfusion in isolated rat heart

It has been recently shown that long-term thyroxine administration increases the tolerance of the heart to ischaemia. The present study investigated whether thyroxine induced cardioprotection involves alterations in the pattern of p38 mitogen activated protein kinase (p38MAPK) and c-Jun NH₂-terminal kinases (JNKs) activation during ischaemia-reperfusion. L-thyroxine (T4) was administered in Wistar rats (25 g/100 g/day, subcutaneously) for 2 weeks (THYR), while normal animals served as controls (NORM). NORM and THYR isolated rat hearts were perfused in Langendorff mode and subjected to 10 or 20 min of zero-flow global ischaemia only and also to 20 min of ischaemia followed by 10, 20 or 45 min of reperfusion. Postischaemic recovery of left ventricular developed pressure at 45 min of reperfusion was expressed as % of the initial value. Activation of p38 MAPK and JNKs was assessed at the different times of the experimental setting by standard Western blotting techniques using a dual phospho p38MAPK and phospho JNKs (p46/p54) antibodies. Activation of p38 MAPK was significantly attenuated during ischaemia and reperfusion in thyroxine treated hearts compared to normal hearts. JNKs were found to be activated only during the reperfusion period. The levels of phospho JNKs were found to be lower in thyroxine treated hearts as compared to untreated hearts, though not at a statistically significant level. Postischaemic functional recovery was higher in THYR as compared to NORM, p < 0.05. In summary, in hearts pretreated with thyroxine, p38 MAPK was attenuated during ischaemia and at reperfusion and this was associated with improved postischaemic recovery of function.     

Amperometric cytochrome c3-based biosensor for chromate determination

The chromate reductase activity of cytochrome c(3) (Cyt c(3), M(r) 13000), isolated from the sulfate-reducing bacterium Desulfomicrobium norvegicum, was used to develop an amperometric biosensor to measure chromate (CrO(4)(2-)) bioavailability. The performance of various biosensor configurations for qualitative and quantitative determination of Cr(VI) was studied. Biosensor properties depend on the technique used to immobilize the enzyme on the electrode (glassy carbon electrode). Immobilization of Cyt c(3) by entrapment in poly 3,4-ethylenedioxythiophene films denatured the enzyme, while application of an adsorption technique did not affect enzyme activity but the detection range was limited. The best results were obtained with dialysis membranes, which allowed the determination of Cr(VI) from 0.20 to 6.84 mg l(-1) (3.85-132 microM) with a sensitivity of 35 nA mg(-1) l (1.82 nA microM(-1)). No interference was observed with As(V), As(III) and Fe(III). Only a small amount of Cyt c(3) (372 ng of protein) was needed for this biosensor.     

DIP: the database of interacting proteins

The Database of Interacting Proteins (DIP; http://dip.doe-mbi.ucla.edu) is a database that documents experimentally determined protein-protein interactions. This database is intended to provide the scientific community with a comprehensive and integrated tool for browsing and efficiently extracting information about protein interactions and interaction networks in biological processes. Beyond cataloging details of protein-protein interactions, the DIP is useful for understanding protein function and protein-protein relationships, studying the properties of networks of interacting proteins, benchmarking predictions of protein-protein interactions, and studying the evolution of protein-protein interactions.     

Circulating Insulin Concentrations,Smoking, and Alcohol Intake Are Important Independent Predictors of Leptin in Young Healthy Men

Objective : Leptin, an adipocyte-secreted hormone, has been shown to signal the status of energy stores to the brain, regulate energy homeostasis, and mediate the neuroendocrine response to food deprivation. Obesity is associated with increased leptin levels, and several hormones, including insulin and glucocorticoids, have been associated with leptin levels and expression in rodents. Although obesity has been strongly associated with increased leptin in humans, a significant percentage of leptin's variability remains unexplained. The role of endogenous hormones, demographic factors, or certain life-style factors in explaining the residual variability of leptin levels has not yet been clarified. We performed this cross-sectional study to document the relative importance of obesity, lifestyle factor, and endogenous hormones in determining serum leptin levels. Research Methods and Procedures : We measured serum concentrations of insulin, Cortisol, testosterone, growth hormone, and dehydroepiandrosterone sulfate; ascertained anthropometric, demographic, and lifestyle characteristics; and studied these variables in relationship to serum leptin concentrations in a sample of young healthy men. Results : Obesity and alcohol intake were independently and positively associated with circulating leptin concentrations. Additionally, cigarette smoking was negatively and independently associated with leptin concentrations. Finally, serum insulin concentration was an independent hormonal determinant of circulating leptin concentrations, whereas serum testosterone was negatively associated with leptin only by bivariate analysis. Discussion : We conclude that, in addition to obesity, cigarette smoking, alcohol intake, and serum insulin levels are associated with leptin levels in a population of healthy young men.     

Resveratrol-Induced Vascular Progenitor Differentiation towards Endothelial Lineage via MiR-21/Akt/β-Catenin Is Protective in Vessel Graft Models

Background and Purpose

Vessel graft failure is typically associated with arteriosclerosis, in which endothelial dysfunction/damage is a key event. Resveratrol has been shown to possess cardioprotective capacity and to reduce atherosclerosis. We aimed to study the influence of resveratrol on the behavior of resident stem cells that may contribute to graft arteriosclerosis.

Experimental Approach

Vascular resident progenitor cells and embryonic stem cells were treated with resveratrol under differentiating conditions and endothelial markers expression was evaluated. Expression of miR-21 and β-catenin was also tested and exogenously modified. Effects of resveratrol treatment in an ex vivo re-endothelialization model and on mice undergone vascular graft were evaluated.

Key Results

Resveratrol induced expression of endothelial markers such as CD31, VE-cadherin and eNOS in both progenitor and stem cells. We demonstrated that resveratrol significantly reduced miR-21 expression, which in turn reduced Akt phosphorylation. This signal cascade diminished the amount of nuclear β-catenin, inducing endothelial marker expression and increasing tube-like formation by progenitor cells. Both the inhibition of miR-21 and the knockdown of β-catenin were able to recapitulate the effect of resveratrol application. Ex vivo, progenitor cells treated with resveratrol produced better endothelialization of the decellularized vessel. Finally, in a mouse model of vessel graft, a resveratrol-enhanced diet was able to reduce lesion formation.

Conclusions and Implications

We provide the first evidence that oral administration of resveratrol can reduce neointimal formation in a model of vascular graft and elucidated the underpinning miR-21/Akt/β-catenin dependent mechanism. These findings may support the beneficial effect of resveratrol supplementation for graft failure prevention.     

Network Reconstruction Based on Proteomic Data and Prior Knowledge of Protein Connectivity Using Graph Theory

Modeling of signal transduction pathways is instrumental for understanding cells’ function. People have been tackling modeling of signaling pathways in order to accurately represent the signaling events inside cells’ biochemical microenvironment in a way meaningful for scientists in a biological field. In this article, we propose a method to interrogate such pathways in order to produce cell-specific signaling models. We integrate available prior knowledge of protein connectivity, in a form of a Prior Knowledge Network (PKN) with phosphoproteomic data to construct predictive models of the protein connectivity of the interrogated cell type. Several computational methodologies focusing on pathways’ logic modeling using optimization formulations or machine learning algorithms have been published on this front over the past few years. Here, we introduce a light and fast approach that uses a breadth-first traversal of the graph to identify the shortest pathways and score proteins in the PKN, fitting the dependencies extracted from the experimental design. The pathways are then combined through a heuristic formulation to produce a final topology handling inconsistencies between the PKN and the experimental scenarios. Our results show that the algorithm we developed is efficient and accurate for the construction of medium and large scale signaling networks. We demonstrate the applicability of the proposed approach by interrogating a manually curated interaction graph model of EGF/TNFA stimulation against made up experimental data. To avoid the possibility of erroneous predictions, we performed a cross-validation analysis. Finally, we validate that the introduced approach generates predictive topologies, comparable to the ILP formulation. Overall, an efficient approach based on graph theory is presented herein to interrogate protein–protein interaction networks and to provide meaningful biological insights.     

Immune Response Gene Expression in Colorectal Cancer Carries Distinct Prognostic Implications According to Tissue,Stage and Site: A Prospective Retrospective Translational Study in the Context of a Hellenic Cooperative Oncology Group Randomised Trial

Background

Although host immune response is an emerging prognostic factor for colorectal cancer, there is no consensus on the optimal methodology, surrogate markers or tissue for study.

Patients and Methods

Tumour blocks were prospectively collected from 344 patients with stage II/III colorectal cancer (CRC) treated with adjuvant chemotherapy. Whole section lymphocytic infiltration was studied along with mRNA expression of CD3Z, CD8, CD4, CXCL9, CXCL13, IGHM, FOXP3, SNAI2 and ESR1 by qRT-qPCR in tissue microarray (TMA) cores from the centre of tumour, invasive margin and adjacent normal mucosa.

Results

Lymphocytic infiltration, deficient MMR (10.9%), KRAS (40.7%) and BRAF (4.9%) mutations or single mRNA gene expression were not prognostic. Tumour ESR1 gene expression (Hazard Ratio [HR] for relapse 2.33, 95% CI 1.35-4.02; HR for death 1.74, 95% CI 1.02-2.97) and absence of necrosis (HR for relapse 1.71, 95% CI 1.05-2.71; HR for death 1.98, 95% CI 1.14-3.43) were adverse prognostic features. We used CD3Z and CD8 expression in order to devise the mRNA-based Immune Score (mIS) and proceeded to partitioning analysis in 267 patients, with age, stage, tumour site (Right vs Left CRC), KRAS mutation and tumour mIS as input factors. Only in patients with stage III right-sided colon cancer, a low immune response was associated with inferior disease-free survival (mIS-low, HR for relapse 2.28, 95% CI 1.05-8.02). No prognostic significance was seen for tumour mIS in any other stage or site of CRC, or for a similar mIS score derived from adjacent normal mucosa. Independent adverse prognostic significance was retained in multivariable analysis for absence of necrosis, tumour ESR1 expression in all patients and low tumour mIS in stage III right-sided CRC.

Conclusions

In localised CRC, mRNA-based CD3Z/CD8 profiling of tumour immune response may have stage, site and tissue-specific prognostic significance, along with ESR1 expression.

Trial Registration

ANZCTR.org.au ACTRN12610000509066