Novel putative drugs and key initiating genes for neurodegenerative disease determined using network-based genetic integrative analysis

Understanding the genetic causes of neurodegenerative disease (ND) can be useful for their prevention and treatment. Among the genetic variations responsible for ND, heritable germline variants have been discovered in genome-wide association studies (GWAS), and nonheritable somatic mutations have been discovered in sequencing projects. Distinguishing the important initiating genes in ND and comparing the importance of heritable and nonheritable genetic variants for treating ND are important challenges. In this study, we analysed GWAS results, somatic mutations and drug targets of ND from large databanks by performing directed network-based analysis considering a randomised network hypothesis testing procedure. A disease-associated biological network was created in the context of the functional interactome, and the nonrandom topological characteristics of directed-edge classes were interpreted. Hierarchical network analysis indicated that drug targets tend to lie upstream of somatic mutations and germline variants. Furthermore, using directed path length information and biological explanations, we provide information on the most important genes in these created node classes and their associated drugs. Finally, we identified nine germline variants overlapping with drug targets for ND, seven somatic mutations close to drug targets from the hierarchical network analysis and six crucial genes in controlling other genes from the network analysis. Based on these findings, some drugs have been proposed for treating ND via drug repurposing. Our results provide new insights into the therapeutic actionability of GWAS results and somatic mutations for ND. The interesting properties of each node class and the existing relationships between them can broaden our knowledge of ND.     

Molecular characterization and functional analyses of ZtWor1, a transcriptional regulator of the fungal wheat pathogen Zymoseptoria tritici

Zymoseptoria tritici causes the major fungal wheat disease septoria tritici blotch, and is increasingly being used as a model for transmission and population genetics, as well as host–pathogen interactions. Here, we study the biological function of ZtWor1, the orthologue of Wor1 in the fungal human pathogen Candida albicans, as a representative of a superfamily of regulatory proteins involved in dimorphic switching. In Z. tritici, this gene is pivotal for pathogenesis, as ZtWor1 mutants were nonpathogenic and complementation restored the wild‐type phenotypes. In planta expression analyses showed that ZtWor1 is up‐regulated during the initiation of colonization and fructification, and regulates candidate effector genes, including one that was discovered after comparative proteome analysis of the Z. tritici wild‐type strain and the ZtWor1 mutant, which was particularly expressed in planta. Cell fusion and anastomosis occur frequently in ZtWor1 mutants, reminiscent of mutants of MgGpb1, the β‐subunit of the heterotrimeric G protein. Comparative expression of ZtWor1 in knock‐out strains of MgGpb1 and MgTpk2, the catalytic subunit of protein kinase A, suggests that ZtWor1 is downstream of the cyclic adenosine monophosphate (cAMP) pathway that is crucial for pathogenesis in many fungal plant pathogens.     

Enhanced accumulation of the isoprostane, 8-epi-PGF2 alpha,in human aortic and pulmonary valves of patients with coronary heart disease

The aim of the present study was to investigate whether the isoprostane 8-epi-PGF2 alpha differently accumulates in semilunar valves of patients suffering from coronary heart disease (CHD, n = 19) as compared to valves from healthy heart donors (controls, n = 6). Sections from isolated aortic and pulmonary valves were analyzed by semiquantitative immunohistochemistry. The 8-epi-PGF2 alpha-content was determined by using a specific radioimmunoassay. The accumulation of 8-epi-PGF2 alpha in both valves was higher in CHD-patients in comparison to controls (Aortic valves: 36.49 +/- 11.26% vs. 15.78 +/- 3.04%; pulmonary valves: 46.79 +/- 9.80% vs. 14.99 +/- 3.57%). The results from the radioimmunoassay revealed comparable findings in both groups (CHD vs. controls: 395.95 +/- 86.09 vs. 139.50 +/- 47.46 pg/mg protein in the aortic valves and 430.47 +/- 76.30 vs. 147.33 +/- 53.84 pg/mg protein in pulmonary valves). Pulmonary valves seem to be more susceptible to oxidative stress than aortic valves as evidenced by a higher accumulation of 8-epi-PGF2 alpha in CHD patients. Considering the data presented in this study, we suggest that 8-epi-PGF2 alpha is a valuable indicator of oxidative injury in human semilunar valves.     

The Importance of Microhabitat for Biodiversity Sampling

Responses to microhabitat are often neglected when ecologists sample animal indicator groups. Microhabitats may be particularly influential in non-passive biodiversity sampling methods, such as baited traps or light traps, and for certain taxonomic groups which respond to fine scale environmental variation, such as insects. Here we test the effects of microhabitat on measures of species diversity, guild structure and biomass of dung beetles, a widely used ecological indicator taxon. We demonstrate that choice of trap placement influences dung beetle functional guild structure and species diversity. We found that locally measured environmental variables were unable to fully explain trap-based differences in species diversity metrics or microhabitat specialism of functional guilds. To compare the effects of habitat degradation on biodiversity across multiple sites, sampling protocols must be standardized and scale-relevant. Our work highlights the importance of considering microhabitat scale responses of indicator taxa and designing robust sampling protocols which account for variation in microhabitats during trap placement. We suggest that this can be achieved either through standardization of microhabitat or through better efforts to record relevant environmental variables that can be incorporated into analyses to account for microhabitat effects. This is especially important when rapidly assessing the consequences of human activity on biodiversity loss and associated ecosystem function and services.     

Gα and Gβ Proteins Regulate the Cyclic AMP Pathway That Is Required for Development and Pathogenicity of the Phytopathogen Mycosphaerella graminicola

We identified and functionally characterized genes encoding three Gα proteins and one Gβ protein in the dimorphic fungal wheat pathogen Mycosphaerella graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Sequence comparisons and phylogenetic analyses showed that MgGPA1 and MgGPA3 are most related to the mammalian Gα_i and Gα_s families, respectively, whereas MgGPA2 is not related to either of these families. On potato dextrose agar (PDA) and in yeast glucose broth (YGB), MgGpa1 mutants produced significantly longer spores than those of the wild type (WT), and these developed into unique fluffy mycelia in the latter medium, indicating that this gene negatively controls filamentation. MgGpa3 mutants showed more pronounced yeast-like growth accompanied with hampered filamentation and secreted a dark-brown pigment into YGB. Germ tubes emerging from spores of MgGpb1 mutants were wavy on water agar and showed a nested type of growth on PDA that was due to hampered filamentation, numerous cell fusions, and increased anastomosis. Intracellular cyclic AMP (cAMP) levels of MgGpb1 and MgGpa3 mutants were decreased, indicating that both genes positively regulate the cAMP pathway, which was confirmed because the WT phenotype was restored by adding cAMP to these mutant cultures. The cAMP levels in MgGpa1 mutants and the WT were not significantly different, suggesting that this gene might be dispensable for cAMP regulation. In planta assays showed that mutants of MgGpa1, MgGpa3, and MgGpb1 are strongly reduced in pathogenicity. We concluded that the heterotrimeric G proteins encoded by MgGpa3 and MgGpb1 regulate the cAMP pathway that is required for development and pathogenicity in M. graminicola.Signal transduction pathways are important for sensing and responding to different environmental stimuli in both lower and higher eukaryotes. The highly conserved heterotrimeric guanine nucleotide-binding proteins (G proteins) belong to a family of regulatory proteins that are crucial for the transduction of signals, which are perceived by a distinct family of cell surface receptors (4). Heterotrimeric G proteins contain three subunits (α, β, and γ) that are linked in the inactive state. Activation of a Gα subunit by a transmembrane receptor leads to exchange of bound GDP with GTP on the Gα subunit, resulting in dissociation of the Gα and the Gβγ dimeric subunits, which can now interact with downstream effectors that subsequently generate changes in cellular responses (for a review, see reference 10).Filamentous fungi have one Gβ- and usually three Gα-encoding genes that belong to three major groups. Encoded proteins in groups I and III are related to the mammalian Gα_i and Gα_s families, respectively, but group II fungal Gα proteins have no mammalian counterpart (1, 4, 14, 22, 33, 53). Interestingly, the corn smut fungus Ustilago maydis contains a unique fourth Gα-encoding gene, and Saccharomyces cerevisiae contains only two Gα proteins (10, 57). Irrespective of the observed numerical variation, Gα proteins regulate a variety of cellular and developmental responses (4). For plant-pathogenic fungi, Gβ-encoding genes have been characterized functionally (9, 14, 22, 27, 31, 48, 52). Apart from the fact that individual Gα-encoding genes and the Gβ-encoding gene have been demonstrated to regulate growth, reproduction, and virulence, comparative functional characterization of all Gα-encoding genes has been reported only for a few plant-pathogenic fungi, including Magnaporthe grisea, Cryphonectria parasitica, and U. maydis (5, 41, 57).Mycosphaerella graminicola (anamorph Septoria tritici) causes septoria tritici blotch disease in bread and durum wheat in areas with high rainfall during the growing season, particularly in Western Europe, where it is considered to be the most important wheat disease (30). It is a ubiquitous phytopathogen with a lifestyle completely different from that of the aforementioned plant-pathogenic fungi. It is a dimorphic pathogen, and therefore the transition from a yeast-like to a filamentous form is important for initiation of infection (45). M. graminicola does not form appressoria but penetrates the leaves through stomata without forming specific infection structures. Furthermore, as a hemibiotroph, it has a biotrophic phase of about 10 days that is followed by a rapid switch to necrotrophy. The necrotic foliar lesions bear anamorphic and teleomorphic fructifications. M. graminicola is the model fungus for the Mycosphaerellaceae and even for the order Dothideales, an extremely large and diverse class of fungi with over 1,000 named species, including major plant pathogens such as the banana leaf streak fungus Mycosphaerella fijiensis (12, 21). Large expressed sequence tag (EST) libraries and the recently released genome sequence have been instrumental for the identification and characterization of genes involved in the development and pathogenicity of M. graminicola (http://genome.jgi-psf.org/Mycgr3/Mycgr3.home.html). Recently, we reported that genes encoding mitogen-activated protein kinases (MAPKs) (MgFus3, MgSlt2, and MgHog1) and the catalytic (MgTpk2) and regulatory (MgBcy1) subunits of protein kinase A (PKA) are essential pathogenicity factors and regulate specific steps during the infection process (8, 43-45). To extend our knowledge about the role of G proteins in the development and pathogenicity of M. graminicola, we functionally analyzed three Gα-encoding genes and one Gβ-encoding gene of M. graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Our results show the requirement of MgGpa1, MgGpa3, and MgGpb1 for pathogenicity, whereas the latter also negatively regulates cell fusion and anastomosis. Among the G protein-encoding genes characterized in this study, MgGpa3 and MgGpb1 positively regulate the cyclic AMP (cAMP) pathway. MgGpa1 seems to be dispensable for cAMP regulation, whereas MgGpa2 appears to be redundant, for none of the assays rendered altered phenotypes. Our results open new perspectives for studying the regulatory machinery of the cAMP pathway in M. graminicola and other plant-pathogenic fungi.     

Molecular characterization of the wild relatives of wheat using CAAT-box derived polymorphism

A molecular assessment of genetic diversity was performed on a set of 228 selected accessions belonging to the different Aegilops and Triticum species using CAAT-box derived polymorphism (CBDP) markers. Fifteen CBDP primers generated 141 polymorphic fragments with an average of 9.40 per primer. The average of polymorphic information content and resolving power revealed a high efficiency of CBDP markers in analyzing genetic diversity among different wheat genotypes. The diversity indexes including polymorphic loci percent, number of observed and effective alleles, Shannon’s index and gene diversity among different populations were 76.24%, 1.67, 1.49, 0.42 and 0.28, respectively. These essentially coincided with the analysis of molecular variance results, indicating that 86, 68, and 59.46% of genetic variation were found within two Aegilops and Triticum genera and their populations, respectively. Genetic relationships inferred from cluster analysis was matched with STRUCTURE analysis, disclosing the accessions were grouped based on their genomic constitution. Furthermore, these results were confirmed by principal coordinate analysis (PCoA). Taken together, our results suggest that CBDP markers will be useful for genetic diversity assessment in the domesticated and wild relatives of wheat.