Matapax: an online high-throughput genome-wide association study pipeline

High-throughput sequencing and genotyping methods are dramatically increasing the number of observable genetic intraspecies differences that can be exploited as genetic markers. In addition, automated phenotyping platforms and "omics" profiling technologies further enlarge the set of quantifiable macroscopic and molecular traits at an ever-increasing pace. Combined, both lines of technological advances create unparalleled opportunities to identify candidate gene regions and, ideally, even single genes responsible for observed variations in a particular trait via association studies. However, as of yet, this new potential is not sufficiently matched by enabling software solutions to easily exploit this wealth of genotype/phenotype information. We have developed Matapax, a Web-based platform to address this need. Initially, we built the infrastructure to support association studies in Arabidopsis (Arabidopsis thaliana) based on several genotyping efforts covering up to 1,375 Arabidopsis accessions. Based on the user-supplied trait information, associated single-nucleotide polymorphism markers and single-nucleotide polymorphism-harboring or -neighboring genes are identified using both the GAPIT and EMMA libraries developed for R. Additional interrogation is facilitated by displaying candidate regions and genes in a genome browser and by providing relevant annotation information. In the future, we plan to broaden the scope of organisms to other plant species as more genotype/phenotype information becomes available. Matapax is freely available at http://matapax.mpimp-golm.mpg.de and can be accessed using any internet browser.     

BRCA1-IRIS overexpression promotes formation of aggressive breast cancers

Introduction

Women with HER2⁺ or triple negative/basal-like (TN/BL) breast cancers succumb to their cancer rapidly due, in part to acquired Herceptin resistance and lack of TN/BL-targeted therapies. BRCA1-IRIS is a recently discovered, 1399 residue, BRCA1 locus alternative product, which while sharing 1365 residues with the full-length product of this tumor suppressor gene, BRCA1/p220, it has oncoprotein-like properties. Here, we examine whether BRCA1-IRIS is a valuable treatment target for HER2⁺ and/or TN/BL tumors.

Methodology/Principal Findings

Immunohistochemical staining of large cohort of human breast tumor samples using new monoclonal anti-BRCA1-IRIS antibody, followed by correlation of BRCA1-IRIS expression with that of AKT1, AKT2, p-AKT, survivin and BRCA1/p220, tumor status and age at diagnosis. Generation of subcutaneous tumors in SCID mice using human mammary epithelial (HME) cells overexpressing TERT/LT/BRCA1-IRIS, followed by comparing AKT, survivin, and BRCA1/p220 expression, tumor status and aggressiveness in these tumors to that in tumors developed using TERT/LT/Ras^V12-overexpressing HME cells. Induction of primary and invasive rat mammary tumors using the carcinogen N-methyl-N-nitrosourea (NMU), followed by analysis of rat BRCA1-IRIS and ERα mRNA levels in these tumors.High BRCA1-IRIS expression was detected in the majority of human breast tumors analyzed, which was positively correlated with that of AKT1-, AKT2-, p-AKT-, survivin, but negatively with BRCA1/p220 expression. BRCA1-IRIS-positivity induced high-grade, early onset and metastatic HER2⁺ or TN/BL tumors. TERT/LT/BRCA1-IRIS overexpressing HME cells formed invasive subcutaneous tumors that express high AKT1, AKT2, p-AKT and vimentin, but no CK19, p63 or BRCA1/p220. NMU-induced primary and invasive rat breast cancers expressed high levels of rat BRCA1-IRIS mRNA but low levels of rat ERα mRNA.

Conclusion/Significance

BRCA1-IRIS overexpression triggers aggressive breast tumor formation, especially in patients with HER2⁺ or TN/BL subtypes. We propose that BRCA1-IRIS inhibition may be pursued as a novel therapeutic option to treat these aggressive breast tumor subtypes.     

Chronic rejection pathology after orthotopic lung transplantation in mice: the development of a murine BOS model and its drawbacks

Almost all animal models for chronic rejection (CR) after lung transplantation (LTx) fail to resemble the human situation. It was our attempt to develop a representative model of CR in mice. Orthotopic LTx was performed in allografts receiving daily immunosuppression with steroids and cyclosporine. Controls included isografts and mice only undergoing thoracotomy (SHAM). Allografts were sacrificed 2, 4, 6, 8, 10 or 12 weeks after LTx. Pulmonary function was measured repeatedly in the 12w allografts, isografts and SHAM mice. Histologically, all allografts demonstrated acute rejection (AR) around the blood vessels and airways two weeks after LTx. This decreased to 50-75% up to 10 weeks and was absent after 12 weeks. Obliterative bronchiolitis (OB) lesions were observed in 25-50% of the mice from 4-12 weeks. Isografts and lungs of SHAM mice were normal after 12 weeks. Pulmonary function measurements showed a decline in FEV(0.1), TLC and compliance in the allografts postoperatively (2 weeks) with a slow recovery over time. After this initial decline, lung function of allografts increased more than in isografts and SHAM mice indicating that pulmonary function measurement is not a good tool to diagnose CR in a mouse. We conclude that a true model for CR, with clear OB lesions in about one third of the animals, but without a decline in lung function, is possible. This model is an important step forward in the development of an ideal model for CR which will open new perspectives in unraveling CR pathogenesis and exploring new treatment options.     

Macrophage sub-populations and the lipoxin A4 receptor implicate active inflammation during equine tendon repair

Macrophages (Mφ) orchestrate inflammatory and reparatory processes in injured connective tissues but their role during different phases of tendon healing is not known. We investigated the contribution of different Mφ subsets in an equine model of naturally occurring tendon injury. Post mortem tissues were harvested from normal (uninjured), sub-acute (3-6 weeks post injury) and chronically injured (>3 months post injury) superficial digital flexor tendons. To determine if inflammation was present in injured tendons, Mφ sub-populations were quantified based on surface antigen expression of CD172a (pan Mφ), CD14(high)CD206(low) (pro-inflammatory M1Mφ), and CD206(high) (anti-inflammatory M2Mφ) to assess potential polarised phenotypes. In addition, the Lipoxin A(4) receptor (FPR2/ALX) was used as marker for resolving inflammation. Normal tendons were negative for both Mφ and FPR2/ALX. In contrast, M1Mφ predominated in sub-acute injury, whereas a potential phenotype-switch to M2Mφ polarity was seen in chronic injury. Furthermore, FPR2/ALX expression by tenocytes was significantly upregulated in sub-acute but not chronic injury. Expression of the FPR2/ALX ligand Annexin A1 was also significantly increased in sub-acute and chronic injuries in contrast to low level expression in normal tendons. The combination of reduced FPR2/ALX expression and persistence of the M2Mφ phenotype in chronic injury suggests a potential mechanism for incomplete resolution of inflammation after tendon injury. To investigate the effect of pro-inflammatory mediators on lipoxin A(4) (LXA(4)) production and FPR2/ALX expression in vitro, normal tendon explants were stimulated with interleukin-1 beta and prostaglandin E(2). Stimulation with either mediator induced LXA(4) release and maximal upregulation of FPR2/ALX expression after 72 hours. Taken together, our data suggests that although tenocytes are capable of mounting a protective mechanism to counteract inflammatory stimuli, this appears to be of insufficient duration and magnitude in natural tendon injury, which may potentiate chronic inflammation and fibrotic repair, as indicated by the presence of M2Mφ.     

The cost-effectiveness of reclassification sampling for prevalence estimation

Background

Typically, a two-phase (double) sampling strategy is employed when classifications are subject to error and there is a gold standard (perfect) classifier available. Two-phase sampling involves classifying the entire sample with an imperfect classifier, and a subset of the sample with the gold-standard.

Methodology/Principal Findings

In this paper we consider an alternative strategy termed reclassification sampling, which involves classifying individuals using the imperfect classifier more than one time. Estimates of sensitivity, specificity and prevalence are provided for reclassification sampling, when either one or two binary classifications of each individual using the imperfect classifier are available. Robustness of estimates and design decisions to model assumptions are considered. Software is provided to compute estimates and provide advice on the optimal sampling strategy.

Conclusions/Significance

Reclassification sampling is shown to be cost-effective (lower standard error of estimates for the same cost) for estimating prevalence as compared to two-phase sampling in many practical situations.     

Proteome analysis of the farnesol-induced stress response in Aspergillus nidulans--The role of a putative dehydrin

The isoprenoid alcohol farnesol represents a quorum-sensing molecule in pathogenic yeasts, but was also shown to inhibit the growth of many filamentous fungi. In order to gain a deeper insight into the antifungal activity of farnesol, we performed 2D-differential gel electrophoretic analysis (2D-DIGE) of Aspergillus nidulans exposed to farnesol. We observed an increased abundance of antioxidative enzymes and proteins involved in protein folding and the ubiquitin-mediated protein degradation. A striking finding was the strong up-regulation of a dehydrin-like protein (DlpA). Expression analyses suggested the involvement of DlpA in the cellular response to oxidative, osmotic and cold stress. In line with these data, we demonstrated that dlpA expression was regulated by the MAP kinase SakA/HogA. The generation of both a dlpA Tet(on) antisense RNA-producing A. nidulans strain (dlpA-inv) and a ΔdlpA deletion mutant indicated a role of DlpA in conidiation and stress resistance of dormant conidia against heat and ROS. Furthermore, the production of the secondary metabolite sterigmatocystin was absent in both strains dlpA-inv and ΔdlpA. Our results demonstrate the complexity of the farnesol-mediated stress response in A. nidulans and describe a farnesol-inducible dehydrin-like protein that contributes to the high tolerance of resting conidia against oxidative and heat stress.     

Microbial and chemical characterization of underwater fresh water springs in the Dead Sea

Due to its extreme salinity and high Mg concentration the Dead Sea is characterized by a very low density of cells most of which are Archaea. We discovered several underwater fresh to brackish water springs in the Dead Sea harboring dense microbial communities. We provide the first characterization of these communities, discuss their possible origin, hydrochemical environment, energetic resources and the putative biogeochemical pathways they are mediating. Pyrosequencing of the 16S rRNA gene and community fingerprinting methods showed that the spring community originates from the Dead Sea sediments and not from the aquifer. Furthermore, it suggested that there is a dense Archaeal community in the shoreline pore water of the lake. Sequences of bacterial sulfate reducers, nitrifiers iron oxidizers and iron reducers were identified as well. Analysis of white and green biofilms suggested that sulfide oxidation through chemolitotrophy and phototrophy is highly significant. Hyperspectral analysis showed a tight association between abundant green sulfur bacteria and cyanobacteria in the green biofilms. Together, our findings show that the Dead Sea floor harbors diverse microbial communities, part of which is not known from other hypersaline environments. Analysis of the water's chemistry shows evidence of microbial activity along the path and suggests that the springs supply nitrogen, phosphorus and organic matter to the microbial communities in the Dead Sea. The underwater springs are a newly recognized water source for the Dead Sea. Their input of microorganisms and nutrients needs to be considered in the assessment of possible impact of dilution events of the lake surface waters, such as those that will occur in the future due to the intended establishment of the Red Sea-Dead Sea water conduit.     

Administration of triclabendazole is safe and effective in controlling fascioliasis in an endemic community of the bolivian altiplano

Background

The Bolivian northern Altiplano is characterized by a high prevalence of Fasciola hepatica infection. In order to assess the feasibility, safety and efficacy of large-scale administration of triclabendazole as an appropriate public health measure to control morbidity associated with fascioliasis, a pilot intervention was implemented in 2008.

Materials and Methods

Schoolchildren from an endemic community were screened for fascioliasis and treated with a single administration of triclabendazole (10 mg/kg). Interviews to assess the occurrence of adverse events were conducted on treatment day, one week later, and one month after treatment. Further parasitological screenings were performed three months after treatment and again two months later (following a further treatment) in order to evaluate the efficacy of the intervention.

Results

Ninety infected children were administered triclabendazole. Adverse events were infrequent and mild. No serious adverse events were reported. Observed cure rates were 77.8% after one treatment and 97.8% after two treatments, while egg reduction rates ranged between 74% and 90.3% after one treatment, and between 84.2% and 99.9% after two treatments. The proportion of high-intensity infections (≥400 epg) decreased from 7.8% to 1.1% after one treatment and to 0% after two treatments.

Conclusion

Administration of triclabendazole is a feasible, safe and efficacious public health intervention in an endemic community in the Bolivian Altiplano, suggesting that preventive chemotherapy can be applied to control of fascioliasis. Further investigations are needed to define the most appropriate frequency of treatment.