Activated p53 in the anti-apoptotic milieu of tuberous sclerosis gene mutation induced diseases leads to cell death if thioredoxin reductase is inhibited

Apoptosis - Tuberous sclerosis, angiomyolipoma and lymphangioleiomyomatosis are a group of diseases characterized by mutation in tuberous sclerosis genes (TSC 1-2). TSC mutation leads to continuous...     

Shifts in Antarctic megabenthic structure after ice-shelf disintegration in the Larsen area east of the Antarctic Peninsula

The aim of this study was to contribute to a general understanding of the response of the Antarctic macrobenthos to environmental variability and climate-induced changes. The change in population size of selected macrobenthic organisms was investigated in the Larsen A area east of the Antarctic Peninsula in 2007 and 2011 using ROV-based imaging methods. The results were complemented by data from the Larsen B collected in 2007 to allow a conceptual reconstruction of the environment-driven changes before the period of investigation. Both Larsen areas are characterised by ice-shelf disintegration in 1995 and 2002, respectively, as well as high inter-annual variability in sea-ice cover and oceanographic conditions. In 2007 one ascidian species, Molgula pedunculata, was abundant north and south of the stripe of remaining ice shelf between Larsen A and B. Population densities decreased drastically in the Larsen A between 2007 and 2011, coincident with the decrease in Corella eumyota, another ascidian. Among the ophiuroids, the population of deposit feeders increased, while suspension feeders halved their abundance. Current measurements indicated a northward flow between the Larsen B and Larsen A, suggesting that a major physical forcing on benthic population development comes from the South. The results demonstrate that Antarctic macrobenthic populations can exhibit dramatic population dynamics. Analyses of sea-ice dynamics, salinity, temperature and surprisingly ice-shelf disintegration history, however, did not provide any clear evidence for environmental drivers underlying the apparent changes.     

Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici

Acquired resistance is a threat to antifungal efficacy in medicine and agriculture. The diversity of possible resistance mechanisms and highly adaptive traits of pathogens make it difficult to predict evolutionary outcomes of treatments. We used directed evolution as an approach to assess the resistance risk to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. Fenpicoxamid inhibits complex III of the respiratory chain at the ubiquinone reduction site (Qi site) of the mitochondrially encoded cytochrome b, a different site than the widely used strobilurins which inhibit the same complex at the ubiquinol oxidation site (Q_o site). We identified the G37V change within the cytochrome b Q_i site as the most likely resistance mechanism to be selected in Z. tritici. This change triggered high fenpicoxamid resistance and halved the enzymatic activity of cytochrome b, despite no significant penalty for in vitro growth. We identified negative cross-resistance between isolates harbouring G37V or G143A, a Q_o site change previously selected by strobilurins. Double mutants were less resistant to both QiIs and quinone outside inhibitors compared to single mutants. This work is a proof of concept that experimental evolution can be used to predict adaptation to fungicides and provides new perspectives for the management of QiIs.     

A class III histidine kinase acts as a novel virulence factor in Botrytis cinerea

Filamentous ascomycetes contain large numbers of histidine kinases (HK) that belong to eleven classes. Members of class III from different species were previously shown to be involved in osmoregulation and resistance to dicarboximide and phenylpyrrole fungicides. We have inactivated the gene encoding the single group III HK, BOS1, in the economically important plant pathogen Botrytis cinerea. BOS1 inactivation had pleiotropic effects on the fungus. Besides the expected osmosensitivity and resistance to fungicides, null mutants presented additional characteristics indicating that BOS1 is necessary for normal macroconidiation and full virulence. On standard culture media, null mutants very rarely formed conidiophores and those few conidiophores failed to produce conidia. This defect could be partially restored with 1 M sorbitol, suggesting that another BOS1-independent signal cascade may be involved in macroconidiation. The mutants were not found to be hypersensitive to various oxidative stresses but were more resistant to menadione. Finally, pathogenicity tests showed that bos1-null mutants were significantly reduced in the ability to infect host plants. Appressorium morphogenesis was not altered; however, in planta growth was severely reduced. To our knowledge, this is the first class III HK characterized as a pathogenicity factor in a plant-pathogenic ascomycete.     

The allele-specific probe and primer amplification assay, a new real-time PCR method for fine quantification of single-nucleotide polymorphisms in pooled DNA

The evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.     

The HOG1-like MAP kinase Sak1 of Botrytis cinerea is negatively regulated by the upstream histidine kinase Bos1 and is not involved in dicarboximide- and phenylpyrrole-resistance

In filamentous ascomycetes, HOG-like signal transduction cascades are involved in the resistance to hyper-osmotic conditions and to dicarboximides and phenylpyrroles. The histidine kinase (HK) Bos1 and the mitogen-activated protein kinase (MAPK) Sak1 are important for the adaptation to hyper-osmotic and oxidative stress, development, and pathogenicity in the phytopathogenic fungus Botrytis cinerea. However, bos1Δ and sak1Δ mutants created previously, also presented different phenotypes, especially the sak1Δ mutants were not resistant to high fungicide concentrations. Since both single mutants were constructed in different parental strains, phenotypic variations due to the genetic background might be suspected. In order to establish the relationship between both protein kinases, we analyzed Sak1 phosphorylation under the control of the Bos1 HK and we realized epistasis analysis between bos1Δ and sak1Δ mutations through the construction of isogenic single and double mutants. Our results show that Bos1 negatively regulates Sak1 phosphorylation and that Bos1 regulates certain phenotypes independently of Sak1. They include fungicide susceptibility, adaptation and conidiation on high neutral osmolarity.     

Comparison of Predictive Immunohistochemical Marker Expression of Primary Breast Cancer and Paired Distant Metastasis using Surgical Material: A Practice-Based Study

Parallel studies of primary breast carcinomas and corresponding distant metastases samples reveal considerable differences. Our aim was to highlight this issue from another perspective and provide further data based on 98 patient samples: 69 primary breast carcinoma and 85 distant metastases from bone, central nervous system (CNS) and lung (56 paired). Two independent series of immunohistochemical reactions with different antibodies for estrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (Her2), along with HER2 fluroscence in situ hybridization (FISH) were performed on tissue microarrays to classify breast carcinoma and distant metastases samples into Luminal A, Luminal B-proliferating, Luminal B-HER2+, HER2+ and triple negative (TNBC) surrogate breast cancer groups. Correlation and agreement between the two assessments of ER and PgR were fair-to-moderate, and almost perfect for HER2 and Ki67. There was 40% discordance concerning immunophenotype between breast carcinomas and distant metastases. Most common metastatic site of ER+ breast carcinoma was the skeletal system (59.2%), whereas that of TNBCs was the CNS (58.8%) and lungs (23.5%). Distant metastases in bones were mostly luminal (54.3%), in the CNS, Luminal B (53.2%), and in the lung, TNBC (37.5%). The change of drugable properties of primary breast cancers in the respective bone and CNS metastases suggests that characterization of the metastasis is necessary for appropriate treatment planning.