Adaptation strategies of Aspergillus species to environmental challenges

Evolution shaped the adaptability of Aspergilli to diverse stress conditions, such as low temperature or adaptation to hostile environments. We applied different assays and multi-omics approaches to gain deeper understanding about these strategies. The determination of intracellular ATP levels during the freezing process revealed a high resistance of Aspergillus nidulans against cold- and cryostress, suggesting that A. nidulans is able to induce protective mechanisms to withstand environmental stresses. Large-scale proteomics, transcriptomics and metabolomics allowed us to gain insights into the low temperature response of this fungus at 10°C, which revealed an induction of cold protection mechanisms, sexual development and natural product biosynthesis. These findings indicated the existence of a light-independent mechanism to trigger sexual developmental regulated by cold. Further on, the production of so-far uncharacterised secondary metabolites with inhibitory effects against fungi and Gram-positive bacteria was induced, which may represent a valuable source for therapeutic compounds. Another stress is caused by human cells. The adaptation mechanisms of the regulation of extracellular protease secretion by deletion of the transcription factors XprG and PrtT in the human-pathogenic fungus Aspergillus fumigatus were characterised by an LC-MS/MS based proteomic approach. Despite an impaired degradation of substrate proteins, the double deletion strain XprG/PrtT did not show an attenuated virulence in a murine infection model of invasive aspergillosis. Furthermore, XprG and PrtT influenced additional processes, such as cell wall modifications and allergen production. Thus, the mutant strain XprG/PrtT may be useful for investigating the allergic response to fungal protein antigens. In summary, Aspergilli used in this study showed strong adaptability to the applied stress conditions and a valuable multi-omics data set for future studies was generated

The cis-acting CTTC-P1BS module is indicative for gene function of LjVTI12, a Qb-SNARE protein gene that is required for arbuscule formation in Lotus japonicus

Lota F, Wegmueller S, Buer B, et al. The cis-acting CTTC-P1BS module is indicative for gene function of LjVTI12, a Qb-SNARE protein gene that is required for arbuscule formation in Lotus japonicus. The Plant Journal. 2013;74(2):280-293.The majority of land plants live in symbiosis with arbuscular mycorrhizal fungi from the phylum Glomeromycota. This symbiosis improves acquisition of phosphorus (P) by the host plant in exchange for carbohydrates, especially under low-P availability. The symbiosome, constituted by root cortex cells accommodating arbuscular mycorrhizal fungal hyphae, is the site at which bi-directional exchange of nutrients and metabolites takes place. Uptake of orthophosphate (Pi) in the symbiosome is facilitated by mycorrhiza-specific plant Pi transporters. Modifications of the potato Pi transporter 3 (StPT3) promoter were analysed in transgenic mycorrhizal roots, and it was found that the CTTC cis-regulatory element is necessary and sufficient for a transcriptional response to fungal colonization under low-Pi conditions. Phylogenetic foot-printing also revealed binary combination of the CTTC element with the Pi starvation response-associated PHR1-binding site (P1BS) in the promoters of several mycorrhiza-specific Pi transporter genes. Scanning of the Lotus japonicus genome for gene promoters containing both cis-regulatory elements revealed a strong over-representation of genes involved in transport processes. One of these, LjVTI12, encoding a member of the SNARE family of proteins involved in membrane transport, exhibited enhanced transcript levels in Lotus roots colonized with the arbuscular mycorrhizal fungus Glomus intraradices. Down-regulation of LjVTI12 by RNA interference resulted in a mycorrhiza-specific phenotype characterized by distorted arbuscule morphology. The results highlight cooperative cis-regulation which integrates mycorrhiza and Pi starvation signaling with vesicle trafficking in symbiosome development

Self-controlled case series studies: just how rare does a rare non-recurrent outcome need to be?

The self-controlled case series method assumes that adverse outcomes arise according to a non-homogeneous Poisson process. This implies that it is applicable to independent recurrent outcomes. However, the self-controlled case series method may also be applied to unique, non-recurrent outcomes or first outcomes only, in the limit where these become rare. We investigate this rare outcome assumption when the self-controlled case series method is applied to non-recurrent outcomes. We study this requirement analytically and by simulation, and quantify what is meant by ‘rare’ in this context. In simulations we also apply the self-controlled risk interval design, a special case of the self-controlled case series design. To illustrate, we extract data on the incidence rate of some recurrent and non-recurrent outcomes within a defined study population to check whether outcomes are sufficiently rare for the rare outcome assumption to hold when applying the self-controlled case series method to first or unique outcomes. The main findings are that the relative bias should be no more than 5% when the cumulative incidence over total time observed is less than 0.1 per individual. Inclusion of age (or calendar time) effects will further reduce bias. Designs that begin observation with exposure maximise bias, whereas little or no bias will be apparent when there is no time trend in the distribution of exposures, or when exposure is central within time observed

The Somatic Genomic Landscape of Chromophobe Renal Cell Carcinoma

We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) based on multidimensional and comprehensive characterization, including mitochondrial DNA (mtDNA) and whole genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared to other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT up-regulation in cancer distinct from previously-observed amplifications and point mutations

Assessment of Unique and Combined Functions of Poly(ADP-Ribose) Polymerases by Using RNA Interference

Poly(ADP-ribose) polymerases (PARP) catalyze the synthesis of poly (ADP-ribose) (PAR), a reversible modification of proteins, using NAD+ as a substrate. Poly(ADP-ribosyl)ation produced by PARP-1 and PARP-2 is involved in cytoplasmic and nuclear processes, such as chromatin remodeling, DNA damage signaling and repair, RNA processing, and regulation of cell death. Genetic knockout mouse models of PARP-1 and PARP-2 have revealed a degree of redundancy in cellular PARP functions. However, advancements in elucidating this redundancy have been hindered by the embryonic lethality of the combined PARP-1 and PARP-2 genetic knockout in mice. To date there are several in vitro studies on the cellular depletion of PARP-1 and PARP-2, but these reports did neither aim to investigate this degree of redundancy nor try to provide detailed understanding of the consequences of a combined knockdown of PARP-1 and PARP-2.In the present work, a first systematic study on the unique and combined functions of PARP-1 and PARP-2 was provided by RNA interference of both proteins in two different cellular approaches. The first approach using a doxycycline inducible microRNA-adapted shRNA (shRNAmir) system had revealed design difficulties in the expression of a polycistronic head to tail configuration to achieve concurrent expression of two shRNAmir sequences, a design formerly reported to be successful but also problematic in some instances. Here, expression of only the second shRNAmir sequence (i.e. PARP-1) was successful, whereas PARP-2 shRNAmir expression could not be demonstrated in stable PARP-1 and PARP-2 shRNAmir expressing HeLa S3 clonal cell populations. Thus, this first approach using concurrent expression of two shRNAmir sequences was not successful in generating a combined knockdown of PARP-1 and PARP-2 in a cellular in vitro system. Moreover, the study design also cautioned before use of older published target siRNA sequences (likely to express off-target effects), although being successful in generating a stable PARP-1 shRNAmir HeLa S3 clonal cell populations. Therefore, in an alternative approach, previously observed difficulties were addressed and concurrent knockdown of PARP-1 and PARP-2 was performed in transient siRNA transfections in two different human cancer cell lines.To deplete PARP-1 and PARP-2 protein expression in the alternative approach, new and effective PARP-1 and PARP-2 siRNA were generated for use in transient siRNA transfections. Here, single and combined transfections of PARP-1 and PARP-2 siRNA demonstrated a strong knockdown of PARP-1 and/or PARP-2 protein expression in western blot analysis and quantifications of relative mRNA levels in HeLa S3 and U2OS cells. Furthermore, both PARP-1 and PARP 2 siRNA were able to strikingly reduce poly(ADP ribose) formation after oxidative stress, demonstrating a functional loss of poly(ADP-ribosyl)ation capacities in cells.In following analyses of population doubling, cell proliferation after genotoxic stress, clonogenic survival, cell death, and finally cell cycle phase distributions in HeLa S3 and U2OS cells, no functional redundancies between PARP-1 and PARP-2 could be observed. In contrast, a novel function of PARP-2 during cellular proliferation in HeLa S3 and U2OS cell lines was demonstrated. Depletion of PARP-2, but not PARP-1, significantly reduced cellular proliferation dependent processes as examined by population doubling, cell proliferation after genotoxic stress and clonogenic survival. Moreover, this new PARP-2 function during cellular proliferation was also independent of oxidative or genotoxic stress and could not be attributed to alterations in cell death. Changes in cell cycle have been found instead to mediate this new PARP-2 function, demonstrating a cell-type and p53 independent G1 phase cell cycle arrest. Finally, this G1 phase cell cycle arrest was shown to be independent of PARP catalytic activity, which might be due the reported function of PARP-2 as a transcriptional repressor of cell cycle related promoters, such as c-MYC, which regulate the G1 phase cell cycle checkpoint.In summary, this first systematic study on unique and combined functions of PARP-1 and PARP-2 demonstrated no functional redundancies of PARP-1 and PARP-2 in the endpoints analyzed. In contrast, a novel catalytic- and PARP-1-independent function of PARP-2 during cellular proliferation was demonstrated within the present work, which might advance understanding of targeting PARP-2 in cancer therapy to suppress tumor growth

Phenotypic and Proteomic Analysis of the Aspergillus fumigatus ΔPrtT, ΔXprG and ΔXprG/ΔPrtT Protease-Deficient Mutants

Aspergillus fumigatus is the most common mold species to cause disease in immunocompromised patients. Infection usually begins when its spores (conidia) are inhaled into the airways, where they germinate, forming hyphae that penetrate and destroy the lungs and disseminate to other organs, leading to high mortality. The ability of hyphae to penetrate the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases that are thought to enhance penetration by degrading host structural barriers. This study explores the role of the A. fumigatus transcription factor XprG in controlling secreted proteolytic activity and fungal virulence. We deleted xprG, alone and in combination with prtT, a transcription factor previously shown to regulate extracellular proteolysis. xprG deletion resulted in abnormal conidiogenesis and formation of lighter colored, more fragile conidia and a moderate reduction in the ability of culture filtrates (CFs) to degrade substrate proteins. Deletion of both xprG and prtT resulted in an additive reduction, generating a mutant strain producing CF with almost no ability to degrade substrate proteins. Detailed proteomic analysis identified numerous secreted proteases regulated by XprG and PrtT, alone and in combination. Interestingly, proteomics also identified reduced levels of secreted cell wall modifying enzymes (glucanases, chitinases) and allergens following deletion of these genes, suggesting they target additional cellular processes. Surprisingly, despite the major alteration in the secretome of the xprG/prtT null mutant, including two to fivefold reductions in the level of 24 proteases, 18 glucanases, 6 chitinases, and 19 allergens, it retained wild-type virulence in murine systemic and pulmonary models of infection. This study highlights the extreme adaptability of A. fumigatus during infection based on extensive gene redundancy

Transmembrane Potential of Red Blood Cells Under Low Ionic Strength Conditions

Background/Aims: In a variety of investigations described in the literature it was not clear to what extent the transmembrane potential red blood cells (RBCs) was changed after the cells have been transferred into low ionic strength (LIS) solutions. Another open question was to find out how fast the transmembrane potential of RBCs in LIS solution will change and which final new equilibrium value will be reached. Methods: The transmembrane potential of human and bovine RBCs was investigated using the potential-sensitive fluorescent dye DIBAC4(3) (bis(1,3-dibutylbarbituric acid) trimethine oxonol) as well as the CCCP (carbonylcyanide-m-chlorophenylhydrazone) method. Results: Under physiological conditions the transmembrane potential was about -10 mV in agreement with literature data. However, when the RBCs were transferred into an isosmotic low ionic strength medium containing sucrose the transmembrane potential increased to +73 mV and +81 mV for human and bovine RBCs, respectively. In case of human RBCs it continuously decreased reaching finally an equilibrium state of -10 mV again after 30 - 60 min. For bovine RBCs the transmembrane potential declined more slowly reaching a value of +72 mV after 30 min. Conclusions: Investigations of parameters of RBCs depending on transmembrane potential cannot be performed with human RBCs in LIS media

Prevention of the degeneration of human dopaminergic neurons in an astrocyte co-culture system allowing endogenous drug metabolism

Background and purpose Few neuropharmacological model systems use human neurons. Moreover, available test systems rarely reflect functional roles of co-cultured glial cells. There is no human in vitro counterpart of the widely used 1-methyl-4-phenyl-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Experimental Approach We generated such a model by growing an intricate network of human dopaminergic neurons on a dense layer of astrocytes. In these co-cultures, MPTP was metabolized to 1-methyl-4-phenyl-pyridinium (MPP+) by the glial cells, and the toxic metabolite was taken up through the dopamine transporter into neurons. Cell viability was measured biochemically and by quantitative neurite imaging, siRNA techniques were also used. Key Results We initially characterized the activation of PARP. As in mouse models, MPTP exposure induced (poly-ADP-ribose) synthesis and neurodegeneration was blocked by PARP inhibitors. Several different putative neuroprotectants were then compared in mono-cultures and co-cultures. Rho kinase inhibitors worked in both models; CEP1347, ascorbic acid or a caspase inhibitor protected mono-cultures from MPP+ toxicity, but did not protect co-cultures, when used alone or in combination. Application of GSSG prevented degeneration in co-cultures, but not in mono-cultures. The surprisingly different pharmacological profiles of the models suggest that the presence of glial cells, and the in situ generation of the toxic metabolite MPP+ within the layered cultures played an important role in neuroprotection.Conclusions and Implications Our new model system is a closer model of human brain tissue than conventional cultures. Its use for screening of candidate neuroprotectants may increase the predictiveness of a test battery

Dynamics of charge separation in the excited-state chemistry of protochlorophyllide

In this study the excited-state dynamics of protochlorophyllide a (PChlide), the substrate of the enzyme protochlorophyllide oxidoreductase, was examined by means of time-resolved absorption anisotropy and magic-angle measurements. The solvent polarity dependent anisotropy data provide strong evidence for the existence of an excited-state with charge-transfer character. A solvent dependency is also observed in the magic-angle kinetics. In a nonpolar environment wave packet oscillations are monitored while they are quenched in a polar solvent. These results show that the excited-state relaxations of PChlide are determined by the nature of the solvent environment. The implications of these findings are discussed with respect to the enzyme catalysed reaction. (C) 2010 Elsevier B. V. All rights reserved