The versatility of the fungal cytochrome P450 monooxygenase system is instrumental in xenobiotic detoxification

Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.     

ERBB4 Promoter Polymorphism Is Associated with Poor Distant Disease-Free Survival in High-Risk Early Breast Cancer

A number of genetic variants have been linked to increased risk of breast cancer. Little is, however, known about the prognostic significance of hereditary factors. Here, we investigated the frequency and prognostic significance of two ERBB4 promoter region variants, −782G>T (rs62626348) and −815A>T (rs62626347), in a cohort of 1010 breast cancer patients. The frequency of nine previously described somatic ERBB4 kinase domain mutations was also analyzed. Clinical material used in the study consisted of samples from the phase III, adjuvant, FinHer breast cancer trial involving 1010 women. Tumor DNA samples were genotyped for ERBB4 variants and somatic mutations using matrix-assisted laser desorption ionization/time of flight mass spectrometry. Paraffin-embedded tumor sections from all patients were immunohistochemically stained for ErbB4 expression. Association of ERBB4 genotype to distant disease-free survival (DDFS) was assessed using Kaplan-Meier and Cox regression analyses. Genotyping was successful for 91–93% of the 1010 samples. Frequencies observed for the ERBB4 variants were 2.5% and 1.3% for −782G>T and −815A>T, respectively. Variant −815A>T was significantly associated with poor survival (HR  = 2.86 [95% CI 1.15–6.67], P = 0.017). In contrast, variant −782G>T was associated with well-differentiated cancer (P = 0.019). Two (0.2%) ERBB4 kinase domain mutations were found, both of which have previously been shown to be functional and promote cancer cell growth in vitro. These data present the germ-line ERBB4 variant −815A>T as a novel prognostic marker in high-risk early breast cancer and indicate the presence of rare but potentially oncogenic somatic ERBB4 mutations in breast cancer.     

Embryo recovery from exercised mares

The effect of exercise on mare reproductive efficiency was evaluated by comparing rates of embryo recovery from mares assigned to either an exercise regimen or a non-exercise (control) regimen. Exercised mares were worked daily for 30 min under average ambient conditions of >30 °C and >50% humidity. Mares were inseminated during estrus and subjected to uterine flush for embryo recovery on d 7 after ovulation for two consecutive cycles. After this, mares were allocated to the opposite group and allowed an estrous cycle without reproductive manipulation; then insemination and uterine flushing were conducted on two more consecutive cycles. Prostaglandin F_2α was administered on the day of uterine flush. Mare rectal temperature increased during exercise from a mean of 38 °C to a mean of 39.9 °C. Mares had ovulations from smaller follicles when exercised than they did under control conditions (39.8 ± 0.5 compared with 41.5 ± 0.5 mm diameter; P < 0.05), and had an increased time from PGF_2α administration to subsequent ovulation (8.47 ± 0.337 compared with 9.27 ± 0.294 d; P < 0.05). Embryo recovery from control mares was 22 of 35 (63%). Fewer embryos were recovered from exercised mares (11 of 32, 34%; P < 0.05). The proportion of embryos classified as Grade 1 tended to be less in exercised than in non-exercised mares (4 of 11, 36% compared with 16 of 22, 73%; P = 0.051). These data indicate that exercising mares in a hot and humid environment are associated with changes in ovarian follicle development and ovulation, and a reduction in embryo recovery.     

Variable responses of old-field perennials to arbuscular mycorrhizal fungi and phosphorus source

If arbuscular mycorrhizal fungi (AMF) promote phosphorus partitioning of plant hosts, they could provide one mechanism for the maintenance of plant community diversity. We investigated whether AMF improved the ability of old field perennials to grow on a range of phosphorus sources and whether AMF facilitated differential performance of plant species on different phosphorus sources (phosphorus niche partitioning). We manipulated form of phosphorus (control versus different inorganic and organic sources) and AM fungal species (control versus four individual AMF species or an AMF community) for five old field perennials grown in a greenhouse in individual culture. Based on biomass after four months of growth, we found no evidence for phosphorus niche partitioning. Rather, we found that effects of AMF varied from parasitic to mutualistic depending on plant species, AMF species, and phosphorus source (significant Plant × Fungus × Phosphorus interaction). Our results suggest that the degree of AMF benefit to a plant host depends not only on AMF species, plant species, and soil phosphorus availability (as has also been found in other work), but can also depend on the form of soil phosphorus. Thus, the position of any AMF species along the mutualism to parasitism continuum may be a complex function of local conditions, and this has implications for understanding plant competitive balance in the field.     

Microbial community analysis by FISH for mathematical modelling of selective enrichment of gel-entrapped nitrifiers obtained from domestic wastewater

Nitrifying activated sludge from natural domestic sewage was entrapped in hydrogel beads, which were subsequently enriched for nitrifiers in a continuous stirred tank reactor (CSTR). Fluorescently labelled, 16S rRNA-targeted oligonucleotide probes specific for ammonia and nitrite oxidisers were used in combination with DAPI staining to monitor the selectivity of the enrichment process. The growth of both nitrifying and heterotrophic bacteria was more pronounced in the periphery of the beads, leading to a biofilm-like stratification of the biomass during the enrichment. Quantitatively, the relative number of nitrifiers increased from 20% immediately after immobilisation up to 64% after 30 days, but decreased again due to extensive heterotrophic growth. These changes were accompanied by an increase in nitrifying activity for about 30 days, whereupon it reached a stable level. This selective enrichment was mathematically modelled by applying finite difference techniques to the diffusion-reaction mass balances of all soluble substrates relevant in the nitrification process. To model biomass growth and spreading, balanced by both decay and detachment at the surface of the beads, the differential methods were combined with a descrete cellular automaton approach. The spatially two-dimensional model was used to calculate radial concentration profiles within a gel bead, as well as to estimate the corresponding total activity of the reactor. Qualitatively, this model could simulate all essential aspects observed experimentally. However, more and better population data as well as independent estimates of decay and hydrolysis rates are needed to refine and verify the quantitative model. In conclusion, even in the absence of an external carbon source and with excess ammonium, it was only possible to obtain a moderate enrichment of nitrifying cells compared to heterotrophs. Under long-term cultivation, the biofilm-like structure developed in the outer gel layers led to a vigorous competition between auto- and heterotrophs for space, and thereby, access to oxygen. FISH analysis in combination with mathematical modelling constitute a suitable toolbox for analysing the population dynamics and biocatalytic performance of such an ecosystem based on lithoautotrophic primary production.     

Establishing the stochastic nature of intracellular calcium oscillations from experimental data

Calcium has been established as a key messenger in both intra- and intercellular signaling. Experimentally observed intracellular calcium responses to different agonists show a variety of behaviors from simple spiking to complex oscillatory regimes. Here we study typical experimental traces of calcium oscillations in hepatocytes obtained in response to phenylephrine and ATP. The traces were analyzed with methods of nonlinear time series analysis in order to determine the stochastic/deterministic nature of the intracellular calcium oscillations. Despite the fact that the oscillations appear, visually, to be deterministic yet perturbed by noise, our analyses provide strong evidence that the measured calcium traces in hepatocytes are prevalently of stochastic nature. In particular, bursting calcium oscillations are temporally correlated Gaussian series distorted by a monotonic, instantaneous, time-independent function, whilst the spiking behavior appears to have a dynamical nonlinear component whereby the overall determinism level is still low. The biological importance of this finding is discussed in relation to the mechanisms incorporated in mathematical models as well as the role of stochasticity and determinism at cellular and tissue levels which resemble typical statistical and thermodynamic effects in physics.