Impact of recently emerged sterol 14{alpha}-demethylase (CYP51) variants of Mycosphaerella graminicola on azole fungicide sensitivity

The progressive decline in the effectiveness of some azole fungicides in controlling Mycosphaerella graminicola, causal agent of the damaging Septoria leaf blotch disease of wheat, has been correlated with the selection and spread in the pathogen population of specific mutations in the M. graminicola CYP51 (MgCYP51) gene encoding the azole target sterol 14α-demethylase. Recent studies have suggested that the emergence of novel MgCYP51 variants, often harboring substitution S524T, has contributed to a decrease in the efficacy of prothioconazole and epoxiconazole, the two currently most effective azole fungicides against M. graminicola. In this study, we establish which amino acid alterations in novel MgCYP51 variants have the greatest impact on azole sensitivity and protein function. We introduced individual and combinations of identified alterations by site-directed mutagenesis and functionally determined their impact on azole sensitivity by expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a regulatable promoter controlling native CYP51 expression. We demonstrate that substitution S524T confers decreased sensitivity to all azoles when introduced alone or in combination with Y461S. In addition, S524T restores the function in S. cerevisiae of MgCYP51 variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of S. cerevisiae transformants expressing recently emerged MgCYP51 variants carrying combinations of alterations D134G, V136A, Y461S, and S524T reveal a substantial impact on sensitivity to the currently most widely used azoles, including epoxiconazole and prothioconazole. Finally, we exploit a recently developed model of the MgCYP51 protein to predict that the substantial structural changes caused by these novel combinations reduce azole interactions with critical residues in the binding cavity, thereby causing resistance.     

Antiviral potency of adenosine analogues: correlation with inhibition of S-adenosylhomocysteine hydrolase

For a series of adenosine analogues a close correlation (r = 0.986) was found between their antiviral potency (against vesicular stomatitis virus) and their inhibitory effects (Ki/Km) on S-adenosylhomocysteine (AdoHcy) hydrolase; thus, in order of increasing inhibitory potency for both virus replication and AdoHcy hydrolase activity: (S)-9-(2,3-dihydroxypropyl)adenine less than (RS)-3-adenin-9-yl-2-hydroxypropanoic acid (isobutyl ester) less than carbocyclic 3-deazaadenosine less than neplanocin A. Our findings point to AdoHcy hydrolase as the target for the broad-spectrum antiviral activity of these adenosine analogues.     

The response of soil solution chemistry in European forests to decreasing acid deposition

Acid deposition arising from sulphur (S) and nitrogen (N) emissions from fossil fuel combustion and agriculture has contributed to the acidification of terrestrial ecosystems in many regions globally. However, in Europe and North America, S deposition has greatly decreased in recent decades due to emissions controls. In this study, we assessed the response of soil solution chemistry in mineral horizons of European forests to these changes. Trends in pH, acid neutralizing capacity (ANC), major ions, total aluminium (Al_tot) and dissolved organic carbon were determined for the period 1995–2012. Plots with at least 10 years of observations from the ICP Forests monitoring network were used. Trends were assessed for the upper mineral soil (10–20 cm, 104 plots) and subsoil (40–80 cm, 162 plots). There was a large decrease in the concentration of sulphate () in soil solution; over a 10‐year period (2000–2010), decreased by 52% at 10–20 cm and 40% at 40–80 cm. Nitrate was unchanged at 10–20 cm but decreased at 40–80 cm. The decrease in acid anions was accompanied by a large and significant decrease in the concentration of the nutrient base cations: calcium, magnesium and potassium (Bc = Ca²⁺ + Mg²⁺ + K⁺) and Al_tot over the entire dataset. The response of soil solution acidity was nonuniform. At 10–20 cm, ANC increased in acid‐sensitive soils (base saturation ≤10%) indicating a recovery, but ANC decreased in soils with base saturation >10%. At 40–80 cm, ANC remained unchanged in acid‐sensitive soils (base saturation ≤20%,  ≤ 4.5) and decreased in better‐buffered soils (base saturation >20%,  > 4.5). In addition, the molar ratio of Bc to Al_tot either did not change or decreased. The results suggest a long‐time lag between emission abatement and changes in soil solution acidity and underline the importance of long‐term monitoring in evaluating ecosystem response to decreases in deposition.     

Brain-corticosteroid hormone dialogue: Slow and persistent

Summary 1. The stress response system is shaped by genetic factors and life experiences, of which the effect of a neonatal life event is among the most persistent. Here we report studies focused on the nature-nurture question using rat lines genetically selected for extreme differences in dopamine phenotype as well as rats exposed as infants to the traumatic experience of maternal deprivation.2. As key to the endocrine and behavioural adaptations occurring in these two animal models the hormone corticosterone (CORT) is considered. The stress hormone exerts slow and persistent genomic control over neuronal activity underlying the stress response system via high affinity mineralocorticoid (MR) and glucocorticoid receptors (GR). This action is exerted in a coordinate manner and involves after stress due to the rising CORT levels progressive activation of both receptor types.3. Short periods of maternal separation (neonatal handling) trigger subsequently enhanced maternal care and sensory stimulation. However, a prolonged period (24h) of depriving the infant of maternal care disrupts the stress hyporesponsive period (SHRP) and causes an inappropriate rise in CORT. During development exposure to CORT and to sensory stimulation has long-lasting consequences for organization of the stress response system.4. We find that these factors embodied by mother-pup interaction are critical for dopamine phenotype, CORT receptor dynamics and neuroendocrine regulation in adult life. The findings provide a conceptual framework to study dopamine-related psychopathology against a background of genetic predisposition, early life events, stress hormones and brain development.     

A histological and immunohistochemical study of the effects of N-acetyl cysteine on retinopathy of prematurity by modifying insulin-like growth factor-1

Retinopathy of prematurity (ROP) is a vasoproliferative disorder that occurs in premature infants and may lead to permanent visual impairment. We investigated both the possible protective role of N-acetyl cysteine (NAC) for preventing ROP and the role of IGF-1 in the disorder. Forty-five newborn rats were divided into three groups. Group 1 was raised in room air as controls. Group 2 was exposed to 60% oxygen for 14 days after birth, then transferred to room air. Group 3 was exposed to the same conditions as group 2, but received intraperitoneal injections of NAC on postnatal days 7–17. After 35 days, both eyes of all rats were processed for histology. Some sections were stained with hematoxylin and eosin to assess structural changes and other sections were immunostained to determine the location of IGF-1. Frozen sections also were prepared and stained for adenosine triphosphatase to detect retinal blood vessels. Compared to the controls, more blood vessels, many of which were abnormal, and increased IGF-1 expression were observed in group 2. In group 3, abnormal blood vessels and IGF-1 expression were less evident. NAC appeared to be an effective vascular-protective agent for ROP by decreasing IGF-1 expression.     

Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies.     

Towards comparable assessment of the soil nutrient status across scales—Review and development of nutrient metrics

Nutrient availability influences virtually every aspect of an ecosystem, and is a critical modifier of ecosystem responses to global change. Although this crucial role of nutrient availability in regulating ecosystem structure and functioning has been widely acknowledged, nutrients are still often neglected in observational and experimental synthesis studies due to difficulties in comparing the nutrient status across sites. In the current study, we explain different nutrient‐related concepts and discuss the potential of soil‐, plant‐ and remote sensing‐based metrics to compare the nutrient status across space. Based on our review and additional analyses on a dataset of European, managed temperate and boreal forests (ICP [International Co‐operative Programme on Assessment and Monitoring of Air Pollution Effects on Forests] Forests dataset), we conclude that the use of plant‐ and remote sensing‐based metrics that rely on tissue stoichiometry is limited due to their strong dependence on species identity. The potential use of other plant‐based metrics such as Ellenberg indicator values and plant‐functional traits is also discussed. We conclude from our analyses and review that soil‐based metrics have the highest potential for successful intersite comparison of the nutrient status. As an example, we used and adjusted a soil‐based metric, previously developed for conifer forests across Sweden, against the same ICP Forests data. We suggest that this adjusted and further adaptable metric, which included the organic carbon concentration in the upper 20 cm of the soil (including the organic fermentation‐humus [FH] layer), the C:N ratio and of the FH layer, can be used as a complementary tool along with other indicators of nutrient availability, to compare the background nutrient status across temperate and boreal forests dominated by spruce, pine or beech. Future collection and provision of harmonized soil data from observational and experimental sites is crucial for further testing and adjusting the metric.     

Conserved sequences in enzymes of the UDP-GlcNAc/MurNAc family are essential in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase

The UDP-GlcNAc/MurNAc family of eukaryotic and prokaryotic enzymes use UDP-GlcNAc or UDP-MurNAc-pentapeptide as donors, dolichol-P or polyprenol-P as acceptors, and generate sugar-P-P-polyisoprenols. A series of six conserved sequences, designated A through F and ranging from 5 to 13 amino acid residues, has been identified in this family. To determine whether these conserved sequences are required for enzyme function, various mutations were examined in hamster UDP- GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT). Scramble mutations of sequences B-F, generated by scrambling the residues within each sequence, demonstrated that each is important in GPT. While E and F scrambles appeared to prevent stable expression of GPT, scrambling of B- D resulted in GPT mutants that could be stably expressed and bound tunicamycin, but lacked enzymatic activity. Further, the C and D scramble mutants had an unexpected sorting defect. Replacement of sequences B-F with prokaryotic counterparts from either the B.subtilis mraY or E.coli rfe genes also affected GPT by preventing expression of the mutant protein (B, F) or inhibiting its enzymatic activity (C-E). For the C-E replacements, no acquisition of acceptor activity for polyprenol-P, the fully unsaturated natural bacterial acceptor, was detected. These studies show that the conserved sequences of the UDP- GlcNAc/MurNAc family are important, and that the eukaryotic and prokaryotic counterparts are not freely interchangeable. Since several mutants were efficiently expressed and bound tunicamycin, yet lacked enzymatic activity, the data are consistent with these sequences having a direct role in product formation.      

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.