The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore, exogenous H₂O₂ (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P = 0.0101). While both mutants exhibited significant sensitivity to both exogenous gliotoxin (P < 0.001) and H₂O₂ (P < 0.01), unexpectedly, exogenous gliotoxin relieved H₂O₂-induced growth inhibition in a dose-dependent manner (0 to 10 μg/ml). Gliotoxin-containing organic extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P < 0.05) the growth of the ΔgliK²⁶⁹³³ deletion mutant. The A. fumigatus ΔgliK²⁶⁹³³ mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396. These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus ΔgliK²⁶⁹³³ mutant than in those of the wild type (P = 0.0024 [fold difference, 24] and P = 0.0003 [fold difference, 9.6], respectively) and were absent from A. fumigatus ΔgliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial extracts of the A. fumigatus ΔgliK²⁶⁹³³ mutant compared to the wild type (P < 0.001). Determination of the gliotoxin uptake rate revealed a significant difference (P = 0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the ΔgliK⁴⁶⁶⁴⁵ mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new insights into gliotoxin functionality in A. fumigatus.     

A macronutrient optimization platform for micropropagation and acclimatization: using turmeric (<Emphasis Type=Italic>Curcuma longa</Emphasis> L.) as a model plant

Tissue culture medium is often overlooked as a factor in plant biotechnology. Most work uses Murashige and Skoog (MS; Physiol Plant in 15:473–497, 1962) inorganic medium formulation, which is not likely optimal for many of the plant systems where it is used. This current study of macronutrient factors simultaneously altered media volume and amount of tissue (plants per vessel), sucrose, nitrogen (as NO₃⁻ and NH₄⁺ ions), and K⁺ in a d-optimal design space with only 55 experimental units (including five true replicates). Meso- and micro-nutrient concentrations were lowered (5% of MS) to determine which elements were most critical to plantlet quality. Plantlet quality was quantified by multiplication in the laboratory and survival and growth in the greenhouse. Plantlets grown at the lowest plant density, the lowest macronutrient concentration (20 mM), and equi-molar proportions of NH₄⁺/K⁺ resulted in the best multiplication ratio and 100% greenhouse survival. Multiplication ratio in vitro and survival in the greenhouse were well correlated with one another. Laboratory dry mass, media use, sucrose use, and the uptake of the macronutrients NO₃⁻, NH₄⁺, and K⁺ were not well correlated with plantlet quality. Plantlets with the greatest uptake of P, Ca, Mg, and Mn had the best multiplication in the laboratory and on subsequent transfer, acclimatized and grew fastest in the greenhouse. Phosphorus was shown to be most depleted in media. This work demonstrates a platform to simultaneously optimize several nutritive components of tissue culture media to produce plantlets that perform well in both laboratory and greenhouse environments. Plant quality was related with factors outside the macronutrient design, and this platform indicated where to expand the experimental space. Fixed, flat-screen presentations revealed less of the response surface than interactive profiles driven by the reader.     

Inhibition of mitochondrial fusion by α‐synuclein is rescued by PINK1, Parkin and DJ‐1

Aggregation of α‐synuclein (αS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of αS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that αS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, αS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age‐dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous αS. In contrast, siRNA‐mediated downregulation of αS results in elongated mitochondria in cell culture. αS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, αS prevents fusion of differently labelled mitochondrial populations. Thus, αS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of αS is rescued by coexpression of PINK1, parkin or DJ‐1 but not the PD‐associated mutations PINK1 G309D and parkin Δ1–79 or by DJ‐1 C106A.     

MicroRNA Profiling in Prostate Cancer - The Diagnostic Potential of Urinary miR-205 and miR-214

Prostate cancer (PCa) is the most common type of cancer in men in the United States, which disproportionately affects African American descents. While metastasis is the most common cause of death among PCa patients, no specific markers have been assigned to severity and ethnic biasness of the disease. MicroRNAs represent a promising new class of biomarkers owing to their inherent stability and resilience. In the present study, we investigated potential miRNAs that can be used as biomarkers and/or therapeutic targets and can provide insight into the severity and ethnic biasness of PCa. PCR array was performed in FFPE PCa tissues (5 Caucasian American and 5 African American) and selected differentially expressed miRNAs were validated by qRT-PCR, in 40 (15 CA and 25 AA) paired PCa and adjacent normal tissues. Significantly deregulated miRNAs were also analyzed in urine samples to explore their potential as non-invasive biomarker for PCa. Out of 8 miRNAs selected for validation from PCR array data, miR-205 (p<0.0001), mir-214 (p<0.0001), miR-221(p<0.001) and miR-99b (p<0.0001) were significantly downregulated in PCa tissues. ROC curve shows that all four miRNAs successfully discriminated between PCa and adjacent normal tissues. MiR-99b showed significant down regulation (p<0.01) in AA PCa tissues as compared to CA PCa tissues and might be related to the aggressiveness associated with AA population. In urine, miR-205 (p<0.05) and miR-214 (p<0.05) were significantly downregulated in PCa patients and can discriminate PCa patients from healthy individuals with 89% sensitivity and 80% specificity. In conclusion, present study showed that miR-205 and miR-214 are downregulated in PCa and may serve as potential non-invasive molecular biomarker for PCa.     

Cytochrome P450cin (CYP176A1) D241N: Investigating the role of the conserved acid in the active site of cytochrome P450s

P450_cin (CYP176A) is a rare bacterial P450 in that contains an asparagine (Asn242) instead of the conserved threonine that almost all other P450s possess that directs oxygen activation by the heme prosthetic group. However, P450_cin does have the neighbouring, conserved acid (Asp241) that is thought to be involved indirectly in the protonation of the dioxygen and affect the lifetime of the ferric-peroxo species produced during oxygen activation. In this study, the P450_cin D241N mutant has been produced and found to be analogous to the P450_cam D251N mutant. P450_cin catalyses the hydroxylation of cineole to give only (1R)-6β-hydroxycineole and is well coupled (NADPH consumed: product produced). The P450_cin D241N mutant also hydroxylated cineole to produce only (1R)-6β-hydroxycineole, was moderately well coupled (31 ± 3%) but a significant reduction in the rate of the reaction (2% as compared to wild type) was observed. Catalytic oxidation of a variety of substrates by D241N P450_cin were used to examine if typical reactions ascribed to the ferric-peroxo species increased as this intermediate is known to be more persistent in the P450_cam D251N mutant. However, little change was observed in the product profiles of each of these substrates between wild type and mutant enzymes and no products consistent with chemistry of the ferric-peroxo species were observed to increase.     

Soil CO2 efflux in uneven-aged managed forests: temporal patterns following harvest and effects of edaphic heterogeneity

Increased nutrient availability can have a large impact in Australian woodland ecosystems, many of which are very poor in nutrients, particularly phosphorus. A study was conducted in an urban Banksia woodland remnant in Perth, southwest Western Australia to test the hypothesis that the soil nutrient status in areas in good condition (GC), poor condition invaded by the perennial grass Ehrharta calycina (PCe), and poor condition invaded by the perennial herb Pelargonium capitatum (PCp), is reflected in the nutrient status of the native and introduced species. Leaf concentrations of P, K, N, Na, Ca, Mg, S, B, Cu, Fe, Mn and Zn of six native (Banksia attenuata, Banksia menziesii, Allocasuarina humilis, Melaleuca systena, Macrozamia fraseri and Conostylis aculeata) and four introduced species (Ehrharta calycina, Pelargonium capitatum, Gladiolus caryophyllaceus and Briza maxima), were measured. Soil pH, electrical conductivity, N (total), P (total), available P, K, S and organic C were assessed beneath all species on all sites. Significantly higher concentrations of soil P (total) and P (HCO₃) were found at PCe and PCp sites than GC sites, while PCp sites also had significantly higher soil concentrations for N (total) and S. Principal Components Analysis of the leaf analyses showed (a) individual species have characteristically different nutrient concentrations; (b) the introduced species Ehrharta calycina and Pelargonium capitatum clustered separately from each other and by vegetation condition. Leaf concentrations of P were significantly (P<0.05) higher, and K and Cu were significantly lower in PCe and PCp sites compared with those at GC sites. Introduced species leaf nutrient concentrations were significantly greater than native species for all nutrients except Mn which was significantly lower, with no differences for Mg and B. The results indicate a key role for P in the Banksia woodland, and we conclude that higher levels of available P at invaded sites are having a detrimental impact on the ecosystem. These results provide new knowledge to enhance conservation practices for the management of the key threatening process of invasion within a biodiversity hot spot. Section Editor: T. Kalapos     

The Influence of Predator Sex on Chemically Mediated Antipredator Response in the Wolf Spider Pardosa milvina (Araneae: Lycosidae)

The wolf spider, Pardosa milvina, reduces activity in the presence of chemical cues (silk and excreta) from a larger predatory wolf spider, Hogna helluo. Hogna is sexually dimorphic in body size and males and females differ in their propensity to attack prey. Consequently, each sex may present different levels of risk to Pardosa. We measured predation risk of Pardosa in the presence of male or female Hogna. We also assessed Pardosa antipredator responses and survival in the presence or absence of previously deposited chemical cues from male or female Hogna. In the absence of predator chemical cues, Pardosa survived significantly longer in the presence of male Hogna compared with female Hogna. We then assessed Pardosa survival in the presence of chemical cues from each Hogna sex by placing Pardosa in containers previously occupied by a female Hogna, a male Hogna, or no Hogna (control). We then introduced a female Hogna into each container and measured predation latency. Pardosa survived significantly longer in the presence of female and male cues compared with the control treatment. Median survival time of Pardosa was over four times longer on substrates with female Hogna cues compared with male cues, but this difference was not statistically significant. We tested Pardosa activity levels in the presence of chemical cues from male or female Hogna. Both Hogna sexes were maintained in separate containers after which we placed an adult female Pardosa in one of the containers or a blank control container. Pardosa significantly decreased activity in the presence of chemical cues from either sex relative to the control. Activity was lowest on substrates with female Hogna cues, but not significantly lower than on substrates with male Hogna cues. Results suggest that chemical information from male or female Hogna significantly reduces Pardosa activity which results in increased survival.     

Human Amnion Epithelial Cells Induced to Express Functional Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis, an autosomal recessive disorder caused by a mutation in a gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), remains a leading cause of childhood respiratory morbidity and mortality. The respiratory consequences of cystic fibrosis include the generation of thick, tenacious mucus that impairs lung clearance, predisposing the individual to repeated and persistent infections, progressive lung damage and shortened lifespan. Currently there is no cure for cystic fibrosis. With this in mind, we investigated the ability of human amnion epithelial cells (hAECs) to express functional CFTR. We found that hAECs formed 3-dimensional structures and expressed the CFTR gene and protein after culture in Small Airway Growth Medium (SAGM). We also observed a polarized CFTR distribution on the membrane of hAECs cultured in SAGM, similar to that observed in polarized airway cells in vivo. Further, hAECs induced to express CFTR possessed functional iodide/chloride (I^−/Cl⁻) ion channels that were inhibited by the CFTR-inhibitor CFTR-172, indicating the presence of functional CFTR ion channels. These data suggest that hAECs may be a promising source for the development of a cellular therapy for cystic fibrosis.     

Genetic determinants for intramuscular fat content and water-holding capacity in mice selected for high muscle mass

Intramuscular fat content and water-holding capacity are important traits in livestock as they influence meat quality, nutritive value of the muscle, and animal health. As a model for livestock, two inbred lines of the Berlin Muscle Mouse population, which had been long-term selected for high muscle mass, were used to identify genomic regions affecting intramuscular fat content and water-holding capacity. The intramuscular fat content of the Musculus longissimus was on average 1.4 times higher in BMMI806 than in BMMI816 mice. This was accompanied by a 1.5 times lower water-holding capacity of the Musculus quadriceps in BMMI816 mice. Linkage analyses with 332 G₃ animals of reciprocal crosses between these two lines revealed quantitative trait loci for intramuscular fat content on chromosome 7 and for water-holding capacity on chromosome 2. In part, the identified loci coincide with syntenic regions in pigs in which genetic effects for the same traits were found. Therefore, these muscle-weight-selected mouse lines and the produced intercross populations are valuable genetic resources to identify genes that could also contribute to meat quality in other species.