The Predicted Secretome of the Plant Pathogenic Fungus Fusarium graminearum: A Refined Comparative Analysis

The fungus Fusarium graminearum forms an intimate association with the host species wheat whilst infecting the floral tissues at anthesis. During the prolonged latent period of infection, extracellular communication between live pathogen and host cells must occur, implying a role for secreted fungal proteins. The wheat cells in contact with fungal hyphae subsequently die and intracellular hyphal colonisation results in the development of visible disease symptoms. Since the original genome annotation analysis was done in 2007, which predicted the secretome using TargetP, the F. graminearum gene call has changed considerably through the combined efforts of the BROAD and MIPS institutes. As a result of the modifications to the genome and the recent findings that suggested a role for secreted proteins in virulence, the F. graminearum secretome was revisited. In the current study, a refined F. graminearum secretome was predicted by combining several bioinformatic approaches. This strategy increased the probability of identifying truly secreted proteins. A secretome of 574 proteins was predicted of which 99% was supported by transcriptional evidence. The function of the annotated and unannotated secreted proteins was explored. The potential role(s) of the annotated proteins including, putative enzymes, phytotoxins and antifungals are discussed. Characterisation of the unannotated proteins included the analysis of Pfam domains and features associated with known fungal effectors, for example, small size, cysteine-rich and containing internal amino acid repeats. A comprehensive comparative genomic analysis involving 57 fungal and oomycete genomes revealed that only a small number of the predicted F. graminearum secreted proteins can be considered to be either species or sequenced strain specific

Analysis of the Maize dicer-like1 Mutant, fuzzy tassel, Implicates MicroRNAs in Anther Maturation and Dehiscence

Sexual reproduction in plants requires development of haploid gametophytes from somatic tissues. Pollen is the male gametophyte and develops within the stamen; defects in the somatic tissues of the stamen and in the male gametophyte itself can result in male sterility. The maize fuzzy tassel (fzt) mutant has a mutation in dicer-like1 (dcl1), which encodes a key enzyme required for microRNA (miRNA) biogenesis. Many miRNAs are reduced in fzt, and fzt mutants exhibit a broad range of developmental defects, including male sterility. To gain further insight into the roles of miRNAs in maize stamen development, we conducted a detailed analysis of the male sterility defects in fzt mutants. Early development was normal in fzt mutant anthers, however fzt anthers arrested in late stages of anther maturation and did not dehisce. A minority of locules in fzt anthers also exhibited anther wall defects. At maturity, very little pollen in fzt anthers was viable or able to germinate. Normal pollen is tricellular at maturity; pollen from fzt anthers included a mixture of unicellular, bicellular, and tricellular pollen. Pollen from normal anthers is loaded with starch before dehiscence, however pollen from fzt anthers failed to accumulate starch. Our results indicate an absolute requirement for miRNAs in the final stages of anther and pollen maturation in maize. Anther wall defects also suggest that miRNAs have key roles earlier in anther development. We discuss candidate miRNAs and pathways that might underlie fzt anther defects, and also note that male sterility in fzt resembles water deficit-induced male sterility, highlighting a possible link between development and stress responses in plants.ECU Open Access Publishing Support Fun

All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells

BACKGROUND & AIMS: Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen. METHODS: Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity. RESULTS: Cell preparation yielded the following cell counts per gram of liver tissue: 2.0+/-0.4x107 hepatocytes, 1.8+/-0.5x106 Kupffer cells, 4.3+/-1.9x105 liver sinusoidal endothelial cells, and 3.2+/-0.5x105 stellate cells. Hepatocytes were identified by albumin (95.5+/-1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5+/-1.2%) and exhibited phagocytic activity, as determined with 1mum latex beads. Endothelial cells were CD146+ (97.8+/-1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of alpha-smooth muscle actin (97.1+/-1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence. CONCLUSIONS: Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease

Bayesian integrated modeling of expression data: a case study on RhoG

Peer reviewe

Impact of metazooplankton on the composition and population dynamics of planktonic ciliates in a shallow, hypertrophic lake.

We conducted an enclosure study in Lake Søbygård, a shallow hypertrophic Danish lake, to examine the impact of metazooplankton on the structure of the microbial food web. Here we present results on ciliate abundance, species composition and trophic interactions during 2 consecutive stages of zooplankton succession. Over a 3 wk period, metazooplankton shifted from dominance of rotifers to cyclopoid copepods and thereafter to cladocerans. On 2 different dates with contrasting zooplankton assemblages we performed enclosure experiments where we compared the population dynamics of ciliates in size-fractionated (80% of total abundance) by 3 small-sized taxa: Urotricha spp., Halteria grandinella and Rimostrombidium brachykinetum, which showed different dynamics in response to metazooplankton. In the first experiment, with dominance of rotifers, zooplankton had only a modest predatory impact on the ciliates, and interactions within the ciliate community were probably more important. Larger, raptorial ciliates (e.g. Monodinium sp., Lagynophrya sp.) seemed to have been the main predators of the small ciliates. Different species-specific responses of ciliates within the same size range were observed. In contrast, the second experiment, with dominance of crustacean zooplankton (cladocerans, copepods), demonstrated a dear top-down control of the whole ciliate community by metazooplankton. Predation is probably the dominating regulating mechanism for ciliate abundance, biomass and species composition in Lake Sobygard. In contrast, food limitation is thought to be of minor importance because of generally high concentrations of edible phytoplankton. This view was also supported in our experiments by very high net growth rates of the dominating ciliate species after predator removal (in the range 1.0 to 2.4 d⁻¹). The study revealed 2 characteristics of hypertrophic lakes: first, zooplankton composition and the resulting predation pattern is the decisive factor for the protozoan community structure, and second, the ciliate community is dominated by high densities of a few small-sized species

Ethene stabilization on Cu(111) by surface roughness

The molecular vibrations of ethene adsorbed on roughened Cu(111) surfaces have been investigated with high resolution electron energy loss spectroscopy and density-functional-theory calculations. The roughness was introduced by sputtering or evaporation of copper, respectively, on the cooled surface. We found stabilization of the ethene layer compared to ethene adsorbed on pristine Cu(111). Furthermore, two new vibrational features observed on the rough surface can be assigned to frustrated translations and rotations of the ethene molecule on surface defects and are indicative of a different binding on the rough surface

Ethene stabilization on Cu(111) by surface roughness

The molecular vibrations of ethene adsorbed on roughened Cu(111) surfaces have been investigated with high resolution electron energy loss spectroscopy and density-functional-theory calculations. The roughness was introduced by sputtering or evaporation of copper, respectively, on the cooled surface. We found stabilization of the ethene layer compared to ethene adsorbed on pristine Cu(111). Furthermore, two new vibrational features observed on the rough surface can be assigned to frustrated translations and rotations of the ethene molecule on surface defects and are indicative of a different binding on the rough surface