text:
The maize pathogen Ustilago maydis has to undergo various morphological transitions for the completion of its sexual life cycle. For example, haploid cells respond to pheromone by forming conjugation tubes that fuse at their tips. The resulting dikaryon grows filamentously, expanding rapidly at the apex and inserting retraction septa at the basal pole. In this review, we present progress on the underlying mechanisms regulating such defined developmental programmes. The key findings of the postgenomic era are as follows: (1) endosomes function not only during receptor recycling, but also as multifunctional transport platforms; (2) a new transcriptional master regulator for pathogenicity is part of an intricate transcriptional network; (3) determinants for uniparental mitochondrial inheritance are encoded at the a2 mating-type locus; (4) microtubule-dependent mRNA transport is important in determining the axis of polarity; and (5) a battery of fungal effectors encoded in gene clusters is crucial for plant infection. Importantly, most processes are tightly controlled at the transcriptional, post-transcriptional and post-translational levels, resulting in a complex regulatory network. This intricate system is crucial for the timing of the correct order of developmental phases. Thus, new insights from all layers of regulation have substantially advanced our understanding of fungal development.
Ustilago maydis is a basidiomycete that infects corn (Fig. 1a) and serves as an excellent model for plant pathogenicity (Banuett, 1995; Nadal et al., 2008; Brefort et al., 2009). However, it is also known to be a delicacy in Central America for hundreds of years and has even found its place in the food industry (Fig. 1b–d). Extensive research over the last couple of decades established this microorganism as a model for a number of important cellular processes such as signalling, transcriptional and post-transcriptional regulation, molecular transport, cell cycle regulation, as well as DNA recombination and repair (Bölker, 2001; Kahmann & Kämper, 2004; Perez-Martin et al., 2006; Feldbrügge et al., 2008; Steinberg & Perez-Martin, 2008; Brefort et al., 2009).
The research on DNA recombination and repair is a superb example documenting how the mechanistic insights gained in U. maydis promote research into human health (Llorente & Modesti, 2009). The foundation for work on DNA recombination was laid by R. Holliday more than 40 years ago. He introduced the concept of the now famous Holliday junction of DNA recombination while studying U. maydis (Holliday, 1964, 2004).
More recent research on DNA repair identified the key factor Brh2, a BRCA2 family protein (Kojic et al., 2002, 2011). Its founding member is a human tumour suppressor encoded by a predisposition gene of hereditary breast cancer. Brh2 from U. maydis catalyses the assembly of active recombinase complexes (Yang et al., 2005) and acts in double-strand repair to reunite broken ends (Mazloum & Holloman, 2009b). In addition, it might even be involved in unconventional strand invasion during the repair of defective replication forks (Mazloum & Holloman, 2009a). Based on these studies, mammalian BRCA2 research has gained tremendous momentum in resolving its molecular function. The protein is now considered a universal recombinase regulator that is conserved from lower to higher eukaryotes (Thorslund & West, 2007; Thorslund et al., 2010).
One of the major breakthroughs for the U. maydis research community was the public release of the genome sequence combined with a profound manual annotation that resulted in high-quality data currently curated in the database MUMDB at MIPS (Kämper et al., 2006; MIPS U. maydis database; and the Munich information centre for protein sequences, respectively; http://mips.helmholtz-muenchen.de/genre/proj/ustilago/). This spurred the application of new approaches such as transcriptome-wide DNA microarrays (Scherer et al., 2006; Zarnack et al., 2008; Heimel et al., 2010b), proteome-wide approaches (Böhmer et al., 2007), gene replacement strategies for efficient knockouts or more sophisticated promoter and gene fusions (Brachmann et al., 2004; Kämper, 2004; Garcia-Pedrajas et al., 2008). Establishing fluorescence proteins such as eGfp, mRfp, mCherry, Yfp, Cfp, photoactivatable Gfp and split-Yfp facilitated comprehensive in vivo localization, colocalization and interaction studies (Steinberg & Perez-Martin, 2008; Heimel et al., 2010a; Schuster et al., 2011b).
In this review, we will use the sexual life cycle of U. maydis as a blue print to describe recent findings focusing on the postgenomic era. Complementary information can be found in earlier reviews (Kahmann & Kämper, 2004; Feldbrügge et al., 2006) and in the special issue of Fungal Genetics & Biology dedicated to U. maydis (Kronstad, 2008). For the sake of clarity, the life cycle is divided into four distinct phases: proliferation of haploid cells, mating, filamentation and plant infection. However, the results described here indicate that key players of the underlying cellular events are often important at several additional stages during the life cycle.
Synopsis of the plant-dependent life cycle
The saprophytic phase can be considered as the default state and starts with meiosis during the germination of diploid teliospores. The resulting haploid cells proliferate by budding (Fig. 1e–g). A prerequisite for infection is the mating of two compatible haploid cells, preferentially on the plant surface (Fig. 2). They recognize each other using a pheromone receptor system that consists of seven-transmembrane domain receptors and small lipopeptide pheromones (pra1/2 and mfa1/2, respectively) encoded at the biallelic a mating-type locus (Fig. 3; Bölker et al., 1992; Spellig et al., 1994; Szabó et al., 2002). Active pheromone signalling results in the formation of conjugation tubes, which orient their growth along the pheromone gradient of the mating partner. Thereby, compatible partners approach each other and fuse at their tips, initiating plasmogamy (Fig. 2; Snetselaar et al., 1996). Characteristic for basidiomycetes, plasmogamy and karyogamy are separated in time (Kruzel & Hull, 2010). A stable dikaryon is formed that grows filamentously with a defined axis of polarity. Hyphae expand at the apical growth cone (Fig. 2) and insert retraction septa at regular intervals at the basal pole. These septa separate the viable compartment from hyphal segments devoid of a cytoplasm, resulting in the formation of regularly spaced empty sections (Lehmler et al., 1997; Steinberg et al., 1998). The nuclei travel to the centre of the cell and are positioned in a defined distance of about 10 μm from each other as well as about 50 μm from the tip (Steinberg et al., 1998; Fuchs et al., 2005).
The developmental switch resulting in hyphal growth is genetically controlled by the action of homeodomain transcription factors encoded as two separate subunits, bWest (bW) and bEast (bE), at the multiallelic b mating-type locus (Kronstad & Leong, 1990; Gillissen et al., 1992). The activity of the transcription factor is elegantly coupled to plasmogamy because it can only function as a heterodimer with subunits derived from different mating partners (e.g. bW1/bE19, bW5/bE8, etc.; Kämper et al., 1995). At present, 19 different b alleles that promote outbreeding are known (J. Kämper, pers. commun.; Barnes et al., 2004).
The dikaryotic filament is the infectious form of the fungus. It grows in close contact with the plant and is able to sense surface signals that trigger the formation of appressoria (Mendoza-Mendoza et al., 2009a; Lanver et al., 2010). These are specialized infection structures that enable the pathogen to enter the plant. Initially, hyphae grow intracellularly by invagination of the plant plasma membrane, establishing a tight interaction zone with colonized host cells (Snetselaar & Mims, 1993; Doehlemann et al., 2008b). At later stages, proliferation also occurs intercellularly and the dikaryotic mycelium grows towards bundle sheets (Doehlemann et al., 2008b). Subsequently, massive proliferation and hyphal fragmentation occurs. This is accompanied by an irregular division of host cells, resulting in the formation of tumours at all aerial parts of the plants. After karyogamy, black diploid teliospores develop within the tumours. Ripe tumours rupture and mature spores are spread by the wind. Under favourable conditions, teliospores germinate and release haploid cells. The life cycle begins anew.
Proliferation of haploid cells and cytokinesis
Saprophytic cells exhibit a defined cylindrical cell shape comparable to Schizosaccharomyces pombe. However, they do not divide by insertion of a central septum, but proliferate by budding like Saccharomyces cerevisiae (Fig. 1e–g). During proliferation, the bud expands by polar tip growth, presumably by actin-mediated secretion of remodelling enzymes and building blocks of the cell wall (Banuett & Herskowitz, 2002; Weber et al., 2006). This notion is supported by the observation that an actin-dependent class V myosin localizes to regions of polar growth and is important for cell morphology (Weber et al., 2003). After the daughter cell reaches a certain size, the nucleus migrates from the centre of the mother into the bud where division occurs (Holliday, 1974; O'Donnell & McLaughlin, 1984). At the mother/bud neck region, the nuclear envelope is striped off, nuclear pore complexes are disassembled and chromosomes are released, enabling an open mitosis that takes place within the daughter cell (Steinberg et al., 2001; Straube et al., 2005; Theisen et al., 2008). This process is most probably regulated by a conserved signalling pathway containing small GTPase Ras3 and dual-function germinal centre kinase Don3 (Straube et al., 2005; Sandrock et al., 2006). Chromosome segregation as well as nuclear movement depend on astral microtubules and involve the molecular motor dynein (Fink et al., 2006), a minus-end-directed motor that is split untypically into two subunits Dyn1 and Dyn2 in U. maydis (Straube et al., 2001).
Nuclear and cytoplasmic microtubule organizing centres (MTOCs, minus-ends of microtubules) nucleate dynamic microtubules that are organized in antiparallel bundles traversing the length of unbudded cells (Straube et al., 2003). During cell division, MTOCs are transported by dynein towards the neck region, where they polarize the microtubule cytoskeleton (Fink & Steinberg, 2006). Growing plus-ends reach into the distal pole of the mother as well as towards the growing pole of the daughter cell (Straube et al., 2003).
One transport function of microtubules is the movement of endosomes containing the t-SNARE-like protein Yup1 (Wedlich-Söldner et al., 2000). These endosomes shuttle bidirectionally along microtubules. Their active transport is mediated by the concerted action of dynein and the plus-end-directed molecular motor Kin3, a Kinesin-3 type motor related to UNC104 and KIF1A from higher eukaryotes (Wedlich-Söldner et al., 2002). Strains carrying a temperature-sensitive mutation in yup1 are altered in cell shape, indicating that yup1-positive endosomes are important for morphology (Wedlich-Söldner et al., 2000). However, inhibition of microtubule function does not cause such morphological alterations (Fuchs et al., 2005), indicating that endosomes might exhibit a microtubule-independent function in regulating morphology during cell growth. Two lines of evidence suggest that endosomal transport is necessary for septation. Firstly, endosomes accumulate at the site of septum formation (Wedlich-Söldner et al., 2000; Schink & Bölker, 2009) and secondly kin3Δ strains are defective in cytokinesis due to aberrant septum formation (Wedlich-Söldner et al., 2002).
To complete the cell division, mother and daughter cells need to form two septa consecutively (Fig. 1e–g). Between these septa, a vacuolar fragmentation zone is formed, defining the location of the breakdown of the connecting cell wall and the physical separation (O'Donnell & McLaughlin, 1984; Banuett & Herskowitz, 2002; Weinzierl et al., 2002).
Mutants in this process were identified by their aberrant donut-shaped colonies on plates, hence their name don1 and don3 (Weinzierl et al., 2002; Feldbrügge et al., 2006). In a liquid medium, don mutants exhibit a distinct cell separation defect. They form tree-like cell clusters due to their failure to form a secondary septum (Weinzierl et al., 2002). Don1 is a guanine nucleotide exchange factor (GEF) that specifically activates the small G-protein Cdc42 (Weinzierl et al., 2002; Mahlert et al., 2006; Hlubek et al., 2008). Don3 is a dual-function germinal centre kinase that acts parallel to the Cdc42 signalling pathway (Weinzierl et al., 2002; Sandrock et al., 2006). Chemical genetics revealed that Don3 kinase triggers the dynamic rearrangement of septins from hourglass-shaped collars into ring-like structures, depending on a contractile actomyosin ring (Böhmer et al., 2008, 2009). Thus, a signalling network containing small G-protein Cdc42 and kinase Don3 is crucial in determining septin and actin-ring organization during secondary septum formation.
Substantial progress has recently been made by connecting components of this signalling network to microtubule-dependent transport (Schink & Bölker, 2009). The authors demonstrate that Don1 is targeted to Yup1-positive endosomes via the specific interaction of the lipid-binding FYVE domain of Don1 with phosphatidylinositol 3-phosphate, a lipid present on these endosomes. Strains expressing a don1 variant with mutations in the FYVE domain are defective in secondary septum formation like kin3Δ strains. The data suggest that the exchange factor Don1 is targeted towards sites of secondary septum formation by microtubule-dependent transport of endosomes. Thereby, local activation of its cognate G-protein Cdc42 results in a signalling cascade orchestrating secondary septum formation and separation (Schink & Bölker, 2009). Apart from the recycling of pheromone receptors (Steinberg, 2007b), this is a new function for these endosomes in U. maydis and fits the general view of endosomes operating as multipurpose platforms (Gould & Lippincott-Schwartz, 2009).
Mating and pheromone signalling
Pheromone signalling determining fungal mating belongs to the best-characterized biological signal transduction phenomena studied in eukaryotes (Elion, 2000; Saito, 2010). The main framework was elucidated in S. cerevisiae and, in comparison with various other fungi such as S. pombe and Cryptococcus neoformans, pheromone signalling proved to be an evolutionarily conserved mechanism (Davey, 1998; Bahn et al., 2007). Key components are G-protein-coupled pheromone receptors that recognize cognate peptide pheromones, resulting in the activation of an evolutionarily conserved MAPK module consisting of three kinases (mitogen-activated protein kinase module; MAPKKK, MAPKK and MAPK). The terminal MAPK phosphorylates, among others, transcription factors that determine pheromone-regulated gene expression. A common feature is that the central MAPK pathways crosstalk with other signalling routes to discern appropriate mating conditions such as the nutritional status (Davey, 1998; Saito, 2010).
In U. maydis mating of two haploid cells is a prerequisite for infection (Fig. 2), a process that is regulated by the a and b mating-type loci. The lipopeptide pheromone of one mating partner is perceived by the cognate receptor of the compatible partner (Bölker et al., 1992; Spellig et al., 1994; Szabó et al., 2002). This elicits an intracellular signal transduction pathway leading to the activation of a defined MAPK module (Fig. 3a; MAPKKK Kpp4, MAPKK Fuz7 and MAPK Kpp2; summarized in Feldbrügge et al., 2006). Direct phosphorylation of the central HMG box transcription factor Prf1 (high mobility group; Hartmann et al., 1996; Müller et al., 1999; Kaffarnik et al., 2003) triggers the expression of a defined set of genes containing pheromone response elements in their regulatory regions (Urban et al., 1996b; Zarnack et al., 2008). Among these are the a mating-type genes, mfa1/2 and pra1/2, enhancing the pheromone response. This feed-forward regulation is further supported by autoactivation of the prf1 gene at the transcriptional level (Fig. 3b; Hartmann et al., 1999).
Genome-wide transcriptional profiling revealed that MAPK regulation of Prf1 is necessary for the expression of a defined subset of pheromone-responsive genes. Analysis of strains expressing Prf1 variants with mutated MAPK sites showed that in the majority of cases, pheromone responsiveness of target genes was completely lost while in the remaining cases regulation was alleviated. Among the latter are genes encoding the key transcription factors for filamentous growth bW/bE as well as Rbf1 (Scherer et al., 2006; Heimel et al., 2010b). Thus, target genes respond differentially to MAPK phosphorylation of Prf1 (Zarnack et al., 2008) and MAPK signalling via Prf1 prepares the main transcriptional cascade that regulates filamentous growth and further pathogenic development (Heimel et al., 2010b; Wahl et al., 2010b).
In addition, the pheromone-responsive MAPK module is involved in the regulation of the pheromone-induced cell cycle arrest in the G2 phase, which is a Prf1-independent process (Fig. 3a; Garcia-Muse et al., 2003). This ensures synchronization of the cell cycle of both nuclei after cell fusion. Pheromone stimulation as well as transcriptional activation of a constitutively active MAPKK triggers reduced expression of several genes encoding cell cycle regulators such as B-type cyclin Clb1 or orthologues of polo kinases (Perez-Martin et al., 2006; Zarnack et al., 2008). It is noteworthy that MAPK signalling also promotes a negative feedback loop by inducing the expression of Rok1, a dual-specificity phosphatase that inactivates MAPK Kpp2 (Fig. 3a; Di Stasio et al., 2009).
Moreover, the MAPK module is responsible for eliciting the formation of conjugation tubes, another Prf1-independent event (Fig. 3a; Müller et al., 2003). During the polar growth of these filaments, the pheromone receptor localizes to their tips. This specific localization as well as receptor turnover by endocytosis might be important for the orientation within the pheromone gradient. Thereby, conjugation tubes of compatible mating partners grow towards each other and fuse at their tips (Snetselaar et al., 1996; Fuchs et al., 2006). After plasmogamy, pheromone signalling is switched off by an active bW/bE heterodimer (Laity et al., 1995; Urban et al., 1996b; Heimel et al., 2010a).
Mating on the surface of the plant is crucial for infection. Thus, cell recognition and fusion are influenced by environmental factors such as nutrient availability, hydroxy fatty acids or hydrophobic surfaces (Hartmann et al., 1999; Mendoza-Mendoza et al., 2009a). Two molecular mechanisms for the underlying signalling integration event have been identified. Firstly, an evolutionarily conserved cAMP signalling pathway that consists of a receptor-coupled heterotrimeric G-protein, adenylate cyclase and protein kinase A (PKA) influences mating (Feldbrügge et al., 2006; Brefort et al., 2009). An important molecular mechanism is the direct phosphorylation of Prf1 by cAMP-activated PKA, a regulatory event that is apparently a prerequisite for pheromone response (Fig. 3a; Kaffarnik et al., 2003). PKA-mediated activation of Prf1 is also sufficient to increase the expression of mating-type genes, indicating that upregulation of the intracellular cAMP level favours mating (Kaffarnik et al., 2003; Zarnack et al., 2008). Crosstalk of the PKA and MAPK signalling is also important at later stages of the pathogenic development (Feldbrügge et al., 2006; Brefort et al., 2009).
Secondly, prf1 expression at the transcriptional level is tightly controlled by various regulatory elements in the promoter region (Fig. 3b). For example, the upstream activating sequence (UAS) element integrates different carbon sources (Hartmann et al., 1999) and the unusual MAPK Crk1 signals via this element (Garrido et al., 2004). In addition, the HMG box transcription factor Rop1 as well as the CCAAT-box-binding protein Hap2 are crucial for the activation of prf1 expression via binding sites in the prf1 promoter (Brefort et al., 2005; Mendoza-Mendoza et al., 2009b). Thus, at least four different transcriptional activators (Prf1, yet unknown UAS-binding protein X, Rop1, Hap2) that integrate potentially different signals converge at the prf1 promoter to accurately control the expression of this key transcription factor (Fig. 3b). This is an excellent example of how a central signalling node can integrate various external and internal cues to determine optimal mating conditions.
Filamentous growth and transcriptional networking
Homeodomain transcription factors constitute crucial players in developmental programmes not only in fungi, but also in plants and animals (Pearson et al., 2005; Hay & Tsiantis, 2010). They often function as master regulators eliciting transcriptional cascades that include additional transcription factors. In U. maydis, the homeodomain transcription factor bW/bE is necessary and sufficient to regulate filamentous growth. This Prf1-activated key regulator connects sexual and pathogenic development. Right after its discovery at the b mating-type locus, it was proposed that, like homeodomain transcription factors from higher eukaryotes, it triggers a defined transcriptional programme essential for development (Kronstad & Leong, 1990; Schulz et al., 1990; Gillissen et al., 1992). However, it turned out to be more difficult than expected to identify the responsible target genes. Only very recently a major breakthrough was achieved with the advent of genome-wide transcriptional profiling (Kahmann & Kämper, 2004; Heimel et al., 2010b; Wahl et al., 2010b).
Initially, it was discovered that the activity of the transcription factor was regulated by heterodimerization of subunits derived from different mating partners (Kämper et al., 1995). This information was used to demonstrate that an active bW/bE heterodimer is sufficient to elicit pathogenic development. Haploid strains were generated that express active versions under the control of their own promoters. These strains are called solopathogenic because they infect plants independent of mating (Bölker et al., 1995).
For the identification of functionally important components of filamentous growth, various comparative methods were applied investigating haploid cells and b-induced filaments. Several genes were identified that exhibited significantly increased expression during filamentous growth, but unfortunately, these were all dispensable for infection (Schauwecker et al., 1995; Wösten et al., 1996; Romeis et al., 2000).
A significant improvement was the generation of haploid strains expressing active bW2/bE1 variants under the control of carbon- or nitrogen-source-regulated promoters. For the first time, this allowed a defined time-resolved monitoring of the underlying regulatory cascade (Brachmann et al., 2001). Using RNA fingerprinting, 10 additional targets could be identified (Brachmann et al., 2001). For example, the involvement of MAPK Kpp6 in the early steps of infection such as plant penetration could be demonstrated (Brachmann et al., 2003).
The same strains were the foundation for applying high-density microarrays covering around 90% of all predicted genes (Heimel et al., 2010b). Performing extensive time-course experiments with carbon- and nitrogen-source-mediated activation of the bW/bE heterodimer resulted in the detection of 345 bW/bE-responsive genes (206 induced and 139 repressed; Heimel et al., 2010b). Global analysis of the respective gene functions revealed that 20 genes involved in cell wall remodelling, such as chitin synthases, exochitinases, chitin deacetylases as well as glucanases, are induced, indicating active remodelling of the fungal cell wall during filamentous growth (Heimel et al., 2010b). A number of clustered genes encoding potential effector proteins for plant interaction were also activated (Kämper et al., 2006; Heimel et al., 2010b). Among the downregulated genes are several encoding cell cycle regulators such as cyclins Cln1, Clb1 and Clb2. For Clb1, it has been shown that repression leads to G2 cell cycle arrest (Garcia-Muse et al., 2004). Apparently, the bW/bE heterodimer is responsible for maintaining the G2 cell cycle arrest until the filament resumes the cell cycle after plant penetration (Perez-Martin et al., 2006).
One of the most rewarding results of this analysis was the identification of the downstream master regulator Rbf1. This zinc finger transcription factor, which is an immediate early target of the bW/bE heterodimer at the transcriptional level, is responsible for the vast majority of the gene expression programme (Fig. 4a; Heimel et al., 2010b). Accordingly, overexpression of Rbf1 is sufficient to trigger filamentous growth. Because it is difficult to envision that hundreds of different bW/bE heterodimers trigger the same transcriptional programme, the presence of such a master regulator was no surprise. Interestingly, rbf1 is also a direct Prf1 target and its expression is pheromone induced via MAPK signalling (Fig. 4b; Zarnack et al., 2008). Thus, during the initial phase of mating, Rbf1 activity must be inhibited by a currently unknown mechanism.
Rbf1 amplifies the transcriptional cascade by the activation of various transcriptional regulators such as Biz1 and Fox1 (Fig. 4a; Heimel et al., 2010b). Biz1 is a zinc finger-containing factor involved in cell cycle regulation and is important for appressoria formation and plant penetration (Flor-Parra et al., 2006). Fox1 regulates the expression of numerous effector proteins that appear to be involved in the suppression of plant defence (Zahiri et al., 2010).
Using a temperature-sensitive mutant, it was demonstrated that the active bW/bE transcription factor is not only needed to establish the transcriptional cascade of pathogenic development, but is also crucial at later stages. Interfering with the activity of bW/bE in planta results in an unusual activation of pheromone signalling, defects in cell cycle synchronization and reduced expression of effector proteins. This aberrant regulation correlates with impaired infection (Wahl et al., 2010b).
Another important direct target of the bW/bE transcription factor is clp1 (Fig. 4b). It encodes a regulator that is required for clamp formation, a special structure needed for nuclear sorting during mitotic division of the dikaryon in planta (Scherer et al., 2006). Recently, the level of complexity was added to by demonstrating that Clp1 interacts with bW and counteracts bW/bE-dependent functions such as G2 cell cycle arrest and filamentous growth (Fig. 4b). Clp1 also interacts with Rbf1 during the repression of pheromone signalling (Heimel et al., 2010a). Furthermore, Clp1 binds Cib1, a bZIP transcription factor required for pathogenic development, whose activity is not regulated at the transcriptional level, but at the level of alternative splicing (Fig. 5b; Heimel et al., 2010a). Thus, besides the simple linear transcription cascade Prf1>bW/bE>Rbf1 (Fig. 4a), there is an underlying complicated network of transcription factors and protein interactions that mediate intensive feedback and feedforward regulation to orchestrate pathogenic development (Fig. 4b).
Filamentous growth and cytoskeletal functions
In general, fungal hyphae exhibit a characteristic growth mode. At their apical pole, a growth cone is responsible for the rapid expansion of membrane and cell wall. In subapical regions, filaments are partitioned by septa forming molecular barriers (Harris, 2006; Steinberg, 2007a; Fischer et al., 2008; Zarnack & Feldbrügge, 2010). Polar growth is mediated by intensive membrane dynamics, i.e. local exo- and endocytosis that is supported by vesicle trafficking. This might be promoted by distinct membrane microdomains at the apical pole that are rich in specific lipids such as sphingolipids and ergosterol (Harris et al., 2005; Steinberg, 2007b). Two juxtaposed macromolecular units are involved in growth cone function: the Spitzenkörper and the polarisome. The latter is a multiprotein complex consisting of landmark proteins, signalling molecules such as small GTPases and formins that nucleate actin cables for short-distance transport (Harris et al., 2005; Sudbery & Court, 2007). The Spitzenkörper is thought to function as a supply centre for exocytotic vesicles (Gierz & Bartnicki-Garcia, 2001; Bartnicki-Garcia et al., 2002), for example, specialized vesicles, so-called chitosomes, transport and export chitin synthases as well as building blocks for cell wall synthesis (Bartnicki-Garcia et al., 2006; Riquelme et al., 2007). Active transport along the actin and microtubule cytoskeleton is mediated by molecular motors for short- and long-distance delivery of vesicles, respectively. This process is streamlined for efficiency and any disturbances lead to reduced growth rates or a complete blockage (Harris, 2006; Steinberg, 2007a).
In U. maydis, a number of recent observations confirm that dikaryotic filaments grow like typical hyphae (Steinberg, 2007a). For example, the landmark protein Spa2 localizes to the tip (Carbo & Perez-Martin, 2008) and sphingolipid biosynthesis is important for polar growth. These lipids are most likely part of membrane microdomains at the hyphal tip (Canovas & Perez-Martin, 2009) and could function to enrich signalling components with membrane anchors such as the small G protein Rac1 (Mahlert et al., 2006). Septins might serve as diffusion barriers organizing these membrane subdomains. Strains carrying corresponding loss-of-function mutations are affected in establishing the axis of polarity, whereas septins are dispensable during the maintenance of filamentous growth (Alvarez-Tabares & Perez-Martin, 2010).
Analysis of eight different chitin synthases revealed that Chs7 was particularly important for polar growth and localizes to the hyphal tip like its related family members Chs5 and Mcs1 (Weber et al., 2006). The chitin synthase Mcs1 contains a unique domain with a similarity to myosin motors (Weber et al., 2006). This domain is not required for motility, but apparently for different functions (Treitschke et al., 2010). Mcs1 might be important in early infection stages. It is conceivable that a defective Mcs1 results in altered cell wall composition. This could trigger the plant's defence response (Treitschke et al., 2010).
Short-distance transport of vesicles that, for example, contain chitin synthases is mediated by the actin cytoskeleton, which is essential for filamentous growth (Fuchs et al., 2005; Steinberg, 2007a). The actin-dependent motor Myo5 is potentially involved in polar growth. Accordingly, the motor accumulates at plus-ends of actin cables at the poles of filaments, and loss of Myo5 results in severe defects during polar growth (Weber et al., 2003; Schuchardt et al., 2005).
Long-distance transport of molecular cargo is mediated by the microtubule cytoskeleton (Steinberg, 2007c). Although microtubules are dispensable for filamentous growth, they are needed to determine the axis of polarity. In their absence, growth is retarded and defects in the insertion of basal retraction septa occur (Fuchs et al., 2005; Steinberg, 2007a). One of the main functions of microtubules during filamentous growth appears to be the bidirectional transport of Yup1-positive vesicles that also colocalize with the endosomal marker Rab5, a small GTPase (Fuchs et al., 2006). Although the knowledge about the function of endosomal shuttling is currently scarce, significant progress has been made in analysing and modelling the mechanism of transport by molecular motors (Steinberg, 2007c; Ashwin et al., 2010; Schuster et al., 2011a). Study of plus-end-binding proteins revealed that microtubules are highly polarized, with plus-ends localizing to both poles (Lenz et al., 2006). Similar to haploid cells, Kin3 and split Dyn1/2 mediate anterograde and retrograde transport of endosomes, respectively (Lenz et al., 2006; Schuster et al., 2011b). Dynein as well as its regulators dynactin and Lis1 accumulate at the plus-ends of microtubules, where they might form a reservoir for retrograde transport (Lenz et al., 2006; Schuster et al., 2011a). Apparently, dynein can be loaded onto endosomes either at plus-ends of microtubules or during its retrograde movement (Schuster et al., 2011a, b). Conventional kinesin Kin1 (Lehmler et al., 1997) is responsible for recycling dynein to the microtubule plus-ends for additional rounds of minus-end-directed transport (Becht et al., 2006; Lenz et al., 2006). Notably, kin1 was initially named kin2 in a study describing kin1 and kin2 encoding a Kinesin-7 and Kinesin-1 type motor, respectively (Lehmler et al., 1997). In order to apply a standardized kinesin nomenclature, kin1 and kin2 were subsequently renamed kin7a and kin1, respectively (Lawrence et al., 2004; Schuchardt et al., 2005).
As mentioned above, knowledge of the biological function of endosomal transport during polar growth is still limited. It has been speculated that these vesicles constitute early endosomes that might be important for endocytotic membrane recycling or act during long-distance signalling to the nucleus (Fuchs et al., 2006; Steinberg, 2007a). However, additional work is required to substantiate these assumptions.
Filamentous growth and post-transcriptional regulation
Post-transcriptional regulation is essential in determining the quantitative and spatiotemporal expression of proteins. Key factors are RNA-binding proteins that function, for example, during the splicing, localization, stability and translation of mRNAs (Dreyfuss et al., 2002). These processes can also be coupled. Localization and translation of mRNAs result, for example, in the defined subcellular localization of the encoded protein, a cellular mechanism that is conserved from bacteria to humans (St Johnston, 2005; Du et al., 2007; Nevo-Dinur et al., 2011). mRNA localization is mainly achieved by active transport along the actin or the microtubule cytoskeleton. In S. cerevisiae, ASH1 mRNA is actively transported from the mother cell towards the distal pole of the daughter cell. Local translation at the pole ensures that the encoded transcription factor specifically localizes to the nucleus of the daughter cell in order to ascertain a distinct transcriptional programme (Jansen, 2001; Zarnack & Feldbrügge, 2007). ASH1 mRNA transport is mediated by two RNA-binding proteins, She2p and She3p, that cooperatively bind RNA elements, so-called zipcodes, in the target mRNA. This is essential for actin-dependent transport mediated by myosin (Böhl et al., 2000; Müller et al., 2011). Recently, it was reported that She3p-mediated ASH1 mRNA transport is also operational in filaments of Candida albicans, most likely to regulate hyphal morphology and invasive growth (Elson et al., 2009).
Microtubule-dependent mRNA transport is mainly studied in higher eukaryotes. Important processes regulated at this level are oocyte and embryo development in Drosophila melanogaster as well as neuronal processes in mammals (St Johnston, 2005; Dahm et al., 2007). Although the RNA-binding proteins and the transported target mRNAs are evolutionarily not conserved, the general concept holds true for all investigated transport processes to date: specific RNA-binding proteins recognize zipcodes forming large mRNPs (mRNA-containing ribonucleoprotein complexes) that are transported along microtubules by kinesin(s) and dynein (Holt & Bullock, 2009).
The importance of such control at the post-transcriptional level is an emerging theme for U. maydis. This additional layer of regulation functions in concert with the extensive regulation at the level of transcription described above (Feldbrügge et al., 2008; Vollmeister & Feldbrügge, 2010). For example, although clp1 is a direct and immediate early target gene of bW/bE, the protein accumulates only at later stages during plant penetration, suggesting translational control (Scherer et al., 2006). Moreover, precise expression of the Clp1 interaction partner Cib1 appears to be regulated by alternative splicing (Heimel et al., 2010a).
Investigating various RNA-binding proteins in U. maydis revealed that two of them, Khd4 and Rrm4, are important for filamentous growth (Becht et al., 2005). The loss of Khd4 causes pleiotropic effects such as altered cell and filament morphology as well as defects in pheromone response and plant infection (Becht et al., 2005). The protein contains at least five potential RNA-binding domains of the KH type (hnRNP K homology domain, KH1-5). It constitutes the founding member of a multi-KH domain family of fungal proteins with representatives in other pathogens such as C. neoformans and C. albicans. Khd4 recognizes the sequence AUACCC via the two central tandem-KH domains (KH3 and KH4; Vollmeister et al., 2009). Interestingly, this motif is enriched in the 3′ untranslated region of mRNAs and a number of transcripts, which exhibit altered expression in khd4Δ strains, contain this binding site. Because most mRNAs show an increased abundance in deletion strains, Khd4 might promote mRNA instability (Vollmeister et al., 2009; Vollmeister & Feldbrügge, 2010). Direct target mRNAs, whose expression is regulated by Khd4 and whose function can be related to the mutant phenotypes, are yet to be identified. To this end, we are currently conducting in vivo UV crosslinking experiments (König et al., 2010).
The deletion of rrm4 causes specific defects during filamentous growth, namely bipolar growth and failure to insert retraction septa (Fig. 5a–c; Becht et al., 2005, 2006). Interestingly, this mutant phenotype is reminiscent of growth defects of strains affected in microtubule functions (Lehmler et al., 1997; Steinberg et al., 1998; Fuchs et al., 2005; Schuchardt et al., 2005; Schuster et al., 2011a). Rrm4 contains three N-terminal RNA recognition motifs and a C-terminal MLLE domain that mediate RNA binding (Becht et al., 2006; König et al., 2007) and protein/protein interactions, respectively (Kozlov et al., 2010). Subcellular localization revealed that Rrm4 shuttles in mRNPs along microtubules (Fig. 5d–e; Supporting Information, Movies S1 and S2). RNA binding as well as the formation of motile mRNPs are essential for protein function (Becht et al., 2006; König et al., 2007). The deletion of kin1 results in the accumulation of Rrm4 at the poles. This is consistent with the hypothesis that split dynein mediates the retrograde transport of mRNPs, because conventional kinesin mediates anterograde transport of split dynein to the plus-ends of microtubules (Becht et al., 2006; S. Baumann, T. Pohlmann & M. Feldbrügge, unpublished data). In vivo UV crosslinking revealed that Rrm4 binds a distinct set of mRNAs that contain a potential CA-rich binding motif (König et al., 2009). These mRNAs encode proteins of cytotopically related groups such as proteins involved in translation, mitochondrial proteins or polarity factors. Important examples are ubi1 and rho3 encoding a natural fusion of ubiquitin with ribosomal protein Rpl40 and a small GTPase, respectively (König et al., 2009). RNA live imaging revealed that these mRNAs are molecular cargos of the Rrm4 transport unit. Remarkably, the CA-rich 3′ UTR of ubi1 functions as a cis-active region in increasing the amount and processivity of trafficking (König et al., 2009). This is a characteristic feature of so-called mRNA zipcodes. Rho3 accumulates at retraction septa, suggesting a regulatory role during this process. These data led to the current model that Rrm4 functions in long-distance transport of mRNAs, a process that is conserved throughout evolution (Fig. 5f–g; Zarnack & Feldbrügge, 2007, 2010). The local translation of Rrm4 target mRNAs appears to be important for the subcellular localization of encoded proteins (Fig. 5f–g). Intriguingly, we could recently show that Rrm4-containing mRNPs are cotransported with Yup1-positive endosomes, suggesting yet another function of microtubule-dependent transport of endosomes (discussed above; S. Baumann, T. Pohlmann & M. Feldbrügge, unpublished data).
Filamentous growth and mitochondrial inheritance
Sexual reproduction in eukaryotes goes along with the combination of nuclear and organelle genomes, such as mitochondrial genomes, in the zygote. This allows recombination events to accelerate evolution. However, a common process in eukaryotic sex is uniparental mitochondrial inheritance, a process that results in the asexual inheritance of the organelle genome. A possible explanation is the avoidance of evolutionary conflicts caused by a heteroplasmic situation. For example, mitochondria with increased replication rates, but decreased functional performance might dominate the population, resulting in disadvantages for the zygote (Partridge & Hurst, 1998; Xu, 2005). Possible mechanisms for uniparental mitochondrial inheritance include unequal size and mitochondrial numbers of gametes (Xu, 2005). An extreme example is human oocytes carrying about 10 000 more mitochondrial genomes than sperm cells, providing for a confident head start of maternal mitochondria in the population. Other mechanisms operating postfusion of the gametes include the interplay with nuclear-encoded genes (Basse, 2010). For example, the mating-type specific homeodomain genes control uniparental inheritance in C. neoformans, suggesting that respective target genes encode proteins that determine uniparental inheritance (Xu, 2005; Yan et al., 2007).
In U. maydis, the inheritance of mitochondria is determined at late stages of filamentous growth when the fungus penetrates the plant. This coincides with the start of cell division of the dikaryon, and mitochondria are passed on to the next cell. Importantly, uniparental mitochondrial inheritance exists in U. maydis and the a2 mitotype is predominantly inherited (Fedler et al., 2009). How is this achieved at the molecular level? Initially, it was observed that the a2 mating-type locus, but not its a1 counterpart contains two genes, lga2 and rga2 (left and right genes of the a2 locus), which are only found in U. maydis and close relatives such as the head smut fungus Sporisorium reilianum (Urban et al., 1996a; Schirawski et al., 2005b). Because Lga2 contains a mitochondrial import signal, it was proposed that it might be involved in uniparental mitochondrial inheritance (Urban et al., 1996a). Work accomplished in the last few years has supported this hypothesis and has shed some light on the underlying mechanism (Basse, 2010).
During mating conjugation tubes fuse, plasmogamy occurs and cellular contents including the mitochondria are mixed (Fig. 2). How is it possible to separate the two mitochondrial populations to ensure uniparental inheritance? The key player is Lga2, a protein that is attached to the outside of the mitochondria (Bortfeld et al., 2004; Mahlert et al., 2009). This protein serves two main functions. Firstly, it inhibits the fusion of the mitochondria (Fig. 6) to prevent the mobilization of homing introns and secondly, Lga2 triggers mitochondrial fragmentation and mtDNA degradation (Bortfeld et al., 2004; Mahlert et al., 2009). Rga2 also localizes to the mitochondria even though a clear targeting signal is missing (Bortfeld et al., 2004). It appears to counteract Lga2 by protecting the mitochondria of the a2 mitotype (Fedler et al., 2009; Basse, 2010).
Based on the expression of these genes, the following model can be proposed (Basse, 2010). Like in all genes of the a locus, lga2 and rga2 expression is activated upon pheromone signalling (Urban et al., 1996b). Thereby, a2 mitotype mitochondria are preloaded with Lga2 and Rga2 before cell fusion. In contrast to mfa1/2 and pra1/2, which are repressed by an active bW/bE heterodimer, lga2 is one of the few directly activated targets of the bW/bE regulator, and rga2 expression is maintained constant (Romeis et al., 2000; Brachmann et al., 2001). Consistently, early during plasmogamy, Lga2 prevents the fusion of mitochondria (Fig. 6) and over time Lga2 eliminates those mitochondria that are not sufficiently protected by Rga2, namely mitochondria of the a1 mitotype (Basse, 2010).
Possible downstream effectors for the two functions of Lga2 are dynamin-related GTPase Dnm1 and the mitochondrial protein Mrb1. Dnm1 is crucial in preventing mitochondrial fusion in an Lga2-dependent manner (Mahlert et al., 2009). Mrb1, a regulator of the p32 family, interacts with Rga2 and appears to counteract the function of Lga2 (Bortfeld et al., 2004; Basse, 2010). This is based on the following observation. While a2mrb1Δ strains fail to infect plants, virulence can be restored by the deletion of lga2. Importantly, dikaryons of a1/a2mrb1Δ strains are impaired in virulence because they are arrested at the early phase of infection when uniparental inheritance should occur (Bortfeld et al., 2004). However, its precise mode of action is currently unclear.
This is now the first glimpse of this process, but as so often, the story is more complex. For example, the dominance of a2 mitotypes is not 100%, indicating additional mechanisms independent of the Lga2/Rga2 system (Bortfeld et al., 2004; Fedler et al., 2009). Moreover, in S. reilianum, there are three mating types: a1, a2 and a3. Lga2 and rga2 are present on a2, but successful mating and potential uniparental mitochondrial inheritance is possible between a1 and a3 (Schirawski et al., 2005b).
Plant infection and fungal effector proteins
For successful infection, mating should take place on the plant surface, such as on the leaves, stems or part of the flower. Therefore, it is not surprising that plant-derived signals stimulate the formation of filaments as well as appressoria (Fig. 7). For example, corn lipids trigger filamentous growth in liquid culture (Klose et al., 2004). Moreover, hydrophobicity acts as an inducer of filamentation and appressorium differentiation on artificial surfaces and the latter is further enhanced by the addition of hydroxy fatty acids. Because both signals are present on the plant surface, they are most likely also operational under natural conditions (Fig. 7; Mendoza-Mendoza et al., 2009a).
Two signalling components, the tetraspan membrane protein Sho1 and its interaction partner the single transmembrane mucin Msb2, appear to be involved in sensing the hydrophobic signal. Accordingly, the deletion of the corresponding genes strongly reduces appressoria formation. Sho1 interacts specifically with MAPK Kpp6, suggesting that Sho1/Msb2 operate upstream of a Kpp6-containing MAPK module during early infection (Lanver et al., 2010).
The perception of host signals is not the only crucial event that is a prerequisite for the development of infection structures on the plant surface. Additional processes involve actin-mediated functions (Berndt et al., 2010) as well as components of the N- and O-glycosylation pathways (Schirawski et al., 2005a; Fernandez-Alvarez et al., 2010). Unfortunately, insights into the underlying mechanisms that influence early infection are currently missing.
Once biotrophic fungi enter the plant, they depend on living material for growth and nutrition, so that many biotrophs form specialized feeding structures termed haustoria (Mendgen & Hahn, 2002). During plant invasion of U. maydis fungal hyphae invaginate the plasma membrane of infected host cells, leading to the formation of a biotrophic interface without extensive feeding structures. Instead, hyphae orient their growth towards vascular bundles, where they might feed on transported sugars (Doehlemann et al., 2008a; Brefort et al., 2009). In line with this, a novel sucrose transporter, Srt1, was recently described as a major component ensuring fungal nutrition (Fig. 7; Wahl et al., 2010a). Interestingly, this transporter has a higher substrate affinity than a comparable plant transporter, suggesting a redirection of sugar flow towards the parasite (Wahl et al., 2010a).
The invasion and proliferation of pathogens within a plant are often accompanied by host recognition, usually triggering defence. Thus, for survival, a successful biotroph should avoid or suppress plant immune responses. Indeed, transcriptional profiling of infected maize indicated that U. maydis is recognized by the plant within the first 24 h after infection as, for example, pathogenesis-related genes are activated. At later stages, however, plant defence appears to be specifically shut down (Fig. 7; Doehlemann et al., 2008a). A possible mechanism would be the interference with plant signalling. Conceivably, jasmonic acid signalling, which appears to be induced in infected tissue, antagonizes salicylic acid signalling, a key component of the plant defence against biotrophs (Doehlemann et al., 2008a).
In accordance with the current views, the fungal pathogen uses secreted effectors to trigger alterations of plant responses during biotrophic growth. Effectors are delivered to the biotrophic interface that constitutes a hotspot for plant–pathogen communication. One important finding that went along with the completion of the genome sequence was the discovery of gene clusters coding for novel secreted proteins that are crucial for infection (Fig. 7; Kämper et al., 2006). In five of 12 cases, the deletion of distinct gene clusters led to disease-associated phenotypes, indicating that these clusters contain virulence factors (Kämper et al., 2006). A common feature is that clustered effector genes exhibit strong transcriptional activation during biotrophic growth (Kämper et al., 2006), and many of these genes with virulence-associated phenotypes are regulated in a bW/bE-dependent manner (Wahl et al., 2010b). Apart from clustered effector genes, there are also single genes and gene families scattered throughout the genome that encode potential effectors (Kämper et al., 2006; Doehlemann et al., 2009; Khrunyk et al., 2010). Currently, the best-studied example is Pep1, an effector that is essential for biotrophic development. pep1 deletion mutants arrest during penetration without causing disease symptoms (Doehlemann et al., 2009). They fail to induce jasmonic acid signalling and do not suppress the plant defence mechanism during the early stage of infection. This is accompanied by an accumulation of toxic substances and papillae formation at the penetration sites (Doehlemann et al., 2009).
Remarkably, it appears that specific effector subsets are important during different stages of pathogenic development, as the block of corresponding mutants differed with respect to the infection stage (Kämper et al., 2006). This finding was recently expanded by providing evidence for organ-specific effector functions. Because plant tissues and organs differ strongly in their physiology, the fungus might adapt to each environment using a special, spatiotemporal-regulated assembly of effectors (Skibbe et al., 2010). Recently, the genome of S. reilianum, a related corn pathogen, was sequenced and annotated (http://mips.helmholtz-muenchen.de/genre/proj/sporisorium; Schirawski et al., 2010). Genome mining revealed that many of the genes for novel secreted proteins found in U. maydis are also present. This is likely due to the fact that they parasitize on the same host. However, most shared effectors are highly divergent, which may be attributed to the differing infection strategies of U. maydis and S. reilianum (Schirawski et al., 2010). In essence, during development in planta U. maydis relies on numerous, thoroughly regulated effectors to induce the host to foster its development.
Our tour following the fungal development of U. maydis revealed quite a number of remarkable recent findings. Highlights are for example (1) the endosomal transport of signalling components during cytokinesis (Schink & Bölker, 2009); (2) the discovery of Rbf1 and the underlying extensive regulation at the transcriptional and post-transcriptional level (Heimel et al., 2010a, b); (3) uniparental mitochondrial inheritance (Fedler et al., 2009); (4) microtubule-dependent mRNA transport (König et al., 2009); and (5) fungal effectors during infection (Kämper et al., 2006; Brefort et al., 2009).
In the future, resolving the underlying molecular mechanisms will be essential in answering the following key questions: how do endosomes function during membrane recycling, signalling and microtubule-dependent transport? How is the expression of transcription factors regulated at the post-transcriptional level? What are the precise molecular functions of effectors?
The results obtained will further advance U. maydis as a simple eukaryotic model for various aspects of basic cellular functions. Within the last few years, a few scientists have spearheaded distinct research areas covering several aspects of cell biology and plant pathogenicity. Now appears to be the right time for expansion.
We acknowledge lab members and Dr V. Göhre for critically reading the manuscript. Our deepest gratitude is due to R. Kellner, Dr H. Leal-Lara, Dr K.M. Sneetselar and Dr C. Basse for images shown in Figs 1a, d, 2 and 6, respectively. Special thanks are due to Drs G. Guerra-Sánchez and J. Pablo Pardo-Vazquez for introducing M.F. to Quesadilla cuitlacoche. We are grateful to Dr R. Kahmann and the Max Planck Institute for Terrestrial Microbiology in Marburg for their generous support. S.B. and T.P. are members of the International Max-Planck Research School for Environmental, Cellular and Molecular Microbiology, C.H. is a member of the HHUD graduate school Molecules of Infection and J.S. participates in the graduate school of the CLIB Graduate Cluster Industrial Biotechnology. Our research was in part financed by grants from the German Science Foundation (DFG Fe448/3 and DFG FOR1334).
Supporting Information
Additional Supporting Information may be found in the online version of this article:
Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.


text:
Click on Thumbnails to see full size images.
Please note that over time links change or are no longer online. This is known as link rot or
link decay.
As such, links may appear that were valid originally, but no longer are.
Images that the Drudge Report links to often expire
after several weeks, consequently we usually only display the original link, not the images
after serveral weeks. You can still click on the link to see if the original image is still available.
DrudgeReportArchive.com © 2017 [DMCA]
CT Inc.
Tag cloud phrases:advisers scares advisers split advisers woo again dinner backers sold biden backers clinton advisers huddles key mounts romney pressure quietly quietly mounts resistance lingers rich using robo advisers romney run run again scares banks split delay using robo woo resistance

subset: commoncrawl


text:
Rupert Boneham knows something about backstabbing and alliance building from his multiple turns on the reality series "Survivor." Well, he'll have to use those skills and more in his next effort, running for Indiana governor.
The 47-year-old Boneham announced Saturday (Oct. 22) that he's vying for the Libertarian Party nomination. He started an exploratory committee for the run in late August.
"I have only one interest: Empowering Hoosiers to give back to their communities. If the government puts up roadblocks, then they should be repealed," Boneham says in a statement.
He continues, "Hoosiers have consistently voted in professional politicians and look at the results. Hoosiers should have a different choice in 2012."
Boneham joins a crowded field of contenders looking to replace outgoing Governor Mitch Daniels who has to leave due to term limits. Other candidates include Republican Rep. Mike Pence and former Democratic Indiana House Speaker John Gregg.
As for Boneham's prospects, the state chairman of the Libertarian Party Sam Goldstein says he's "very excited" about the "Survivor" star joining the fray.
"His nomination would be a great asset to the party and I look forward to working with him and his campaign team should he win the nomination at our convention next spring," Goldstein says in a statement.
For the last 20 years, Boneham has been helping at-risk youth through his own charity.


text:
Junying Die Casting Mould Co., Ltd.
Palabras clave:
Metal, Metales, Herrajes
Zinc Alloy Bicycle Lock Part Supplier Junying Provides Oiled Zinc Bicycle Lock Parts, DIN, BS and JIS Standards, Various Color Available.
Name: Zinc Alloy Bicycle Lock Part.
Material: Zinc Alloy.
Certificate: DIN, BS, JIS.
Surface Treatment: Oiled to Prevent the Paint from Peeling Off .
Color of Sample: Red.
Color Available: According to customers' requirements.
Application: Bicycle, Motorbike, Scooter, Motorcycle, Autocycle, etc.
Origin: Dongguan, Guangzhou, China.
Packing: Carton and Pallet or as your requirements.
Production Capacity: 50,000PCS/Month.
We offer OEM services and accept customized orders.
Imprimir esta página
China Aluminum Alloy Lamp Shade Manufacturer: Aluminum Alloy Die Casting Waterproof Lamp Shade, abrasive blasting and electrophoresis, used under 2
China Zinc Alloy Brake Crank manufacturer: Motorcycle Brake Crank Die Casting, motorcycle Zinc Alloy Brake Crank, Electrolytic Chroming surface.
China Aluminum Alloy Shoes Accessory Manufacturer: Aluminum Alloy Die Casting Shoes Accessory, spouting oil on surface with red, blue and
China Aluminum Alloy Connector Manufacturer: Aluminum Alloy Die Casting Connector, no surface treatment, used to connect mechanical parts.
China Aluminum Alloy Auto Reflection Cup Manufacturer: Aluminum Alloy Die Casting Auto Reflection Cup, Vacuum Plated Surface, used as auto
China 6061-T6 Aluminum Set-Up-Box Die Casting Manufacturer Junying Supplies 6061-T6 Aluminum Set-Up-Box, Black E-Coating, for Military Communication.
China Aluminum Alloy Die Casting Hinge Manufacturer Junying Offers Aluminum Alloy Die Casting Window Hinge, Polished Surface, ISO 9001:2008, Window
China Aluminium Alloy Ceiling Lamp Housing Manufacturer Junying Provides Aluminium Alloy A380 Ceiling Lamp Housing Die Casting, Semi-Finished.
China Aluminum Alloy Telecom Accessories Manufacturer Junying Supplies Aluminum Alloy Die Casting Telecom Accessories, Sand Blasting, Silver Plating.
China Aluminum Alloy Household Furniture Die Casting Manufacturer Junying Supplies Polishing Aluminum Alloy Die Casting for Table Leg.
Precision Mould contract manufacturer Junying Metal Manufacturing Co., ltd. has produced precision mould for customers since 2002.
Zinc Alloy Motorbike Brake Disc Lock Part Provider Junying Offers Anti-rust Brake Disc Lock Parts for Scooters, Motorcycles, Autocycles, Bikes.
Aluminium Light Screw Socket Die Cast Part Manufacturer Junying Offers Light Screw Socket Die Cast Parts for Illumination, American Standard.
ADC12 Aluminium Alloy Lamp Body Die Casting Contract Manufacturer Junying Provides Lamp Body Die Castings, SGS, ISO & RoHS, Electroplating.
Cree su perfil de empresa gratuito
Siga a Hotfrog en:
Compartir en:
Foro de emprendedores Hotfrog


text:
track list
Album Canzoni Popolari, Romane E Napoletane
Label Retro Records
Released May 26, 2008
This site contains content from artists, fans, and writers from around the internet in it's natural form. Such content is not representative of Viacom Media Networks.
©2017 Viacom International Inc. All Rights Reserved. MTV and all related titles and logos are trademarks of Viacom International Inc.


text:
Athena Staik, Ph.D., LMFT
Licensed Marriage and Family Therapist
12866 Harbor Drive, Woodbridge, Virginia 22192
I specialize in working with couples, individuals and families, and integrate neuroscience, proven psychological methods, and recent findings on the brain & intimacy to support clients to break free of fears, addictive patterns and reactivity for lasting change.
Well Marriage Center, Ph.D, Psy.D., LMFT, LPC, LCSW
Marriage Counseling Specialists
6204 Old Franconia Rd, Suite A, Alexandria, Virginia 22310
Marriage counseling is our specialty. We're here to support the possibility that you can save and improve your relationship. Our approach is strengths-based and safe for your marriage. We offer 200+ years of experience helping thousands of couples succeed and thrive!
Kristin Rosenthal, MA, LPC
Licensed Professional Counselor
1707 Belle View Blvd C-1, Alexandria, Virginia 22307
Mount Vernon Family Therapy Associates helps families, couples, adults and children by providing a warm, safe, confidential place to explore their inner worlds, and help them connect more deeply with the outer world. We aim to help people have better relationships now.
Robyn Brickel, M.A., LMFT
Licensed Marriage and Family Therapist
300 North Washington Street; # 305, Alexandria, Virginia 22314
Robyn E. Brickel, M.A., LMFT, LLC is a private practice that specializes in the therapeutic treatment of individuals (adolescents and adults), couples, families and groups. The practice offers treatment and educational services for many life issues and transitions.
Gina Binder, M.A.
Resident in Counseling
9300 Forest Point Circle, Suite 103, Manassas, Virginia 20110
I help men and women discover that change is possible. In a safe, compassionate atmosphere, I help you heal from the inside out, by building on your personal strengths and helping you find new ways to relate to yourself, the world around you, and the people in it.
Linda Ritchie, Ph.D.
Licensed Professional Counselor/Marriage & Family Therapist
3801 N Fairfax Drive Suite 61, Arlington, Virginia 22203
Do you struggle from the "common cold" of mental health -anxiety depression, worry, sadness, or unexplained fatigue? Are you experiencing difficulties in your marriage or relationship? I provide professional result oriented counseling.
Christiana Shao, M.S., M.A., LGPC, NCC
Resident in Counseling
300 N Washington St #102, Falls Church, Virginia 22046
Life is difficult and we all need help sometimes. I believe the best way to deal with our problems is to talk through them in a calm, safe place with a nonjudgemental presence. Using a warm, collaborative approach, we will work to help you make changes you need to cope.
Alicia Munoz, MA, LPC
Licensed Professional Counselor
450 West Broad Street Suite 315, Falls Church, Virginia 22046
Unconscious forces can sometimes keep us from fulfilling of our deepest needs and desires—until we explore and understand them. I help you see challenging situations you face as an invitation to grow by taking steps to create the positive change you want in your life.
Christina Schultz, MA
Resident in Counseling, Supervised by Thomas Lamp, LPC
New Directions Counseling, 150 S. Washington St Suite 303, Falls Church, VA, Falls Church, Virginia 22046
Managing expectations, exercising self-care, building self-identity in the face of uncertainties, developing life meaning, and accepting loss and lack of control, are fundamental to maintaining resilience and relationships.
Sapna Doshi, Ph.D.
Licensed Clinical Psychologist
2200 Wilson Blvd, Suite 412, Arlington, Virginia 22201
Finding the right therapist can sometimes be a daunting task. Call today for a free phone consultation so that together we can determine if my approach to therapy will be a good fit for your needs.


text:
Follow all the action as Real Zaragoza travel away to Real Madrid in the Spanish Liga BBVA on 30-Apr-2011. The match kicks off at 17:00
Real Madrid v Real Zaragoza Live
Source: DSG
Source: DSG
Claudio Ranieri has left Leicester.
Leicester have sacked Claudio Ranieri as they face up to a relegation battle just nine months after their stunning Premier League title.
Leicester's euphoria at winning the league last season has evaporated, with Claudio Ranieri leaving the club.
Manchester United are on the longest run without a penalty of any Premier League club, in contrast to their historical reputation.
The latest gossip and rumours from the newspapers and online:WAYNE ROONEY'S agent is in China in a bid to arrange
The latest gossip and rumours from the newspapers and online:WAYNE ROONEY is ready to quit Manchester United and take up


text:
Not so profound
Workout notes: I had bigger plans but my legs weren't ready. So I did a 10K shuffle in 1:09:42 (the 5.1 mile course plus an out and back down the upper Bradley Park Hill).
Yes, the Marathon Route signs are up; Race day is the 16'th of October (Sunday)
Last week's failure has me hungry for "a finish"; time goal just isn't that important to me now. There was a time when I took a marathon finish for granted; that time was over a long time ago (2009?)
Personal: I thought that I had something profound to say about the moment generating function and the negative binomial distribution. I don't. In any event, I can't talk about it in class until I talk about joint density functions.
I was also pleased with myself when I worked about the old "every vector space has a basis" argument in my head (and mostly got it right) until I realized: "yes, you are SUPPOSED to know this stuff". Oh well. (hint: Zorn's Lemma)
Time to type a boring request.
back in Peoria
Math Fest: decent conference, but too much driving. Traffic and construction added time to the normally 5:45 hour drive. But my Google maps application helped me around one traffic jam on the way home. I did get some ideas to consider though.
Workout notes: 3.12 miles on the hotel treadmill in 30 minutes; 11:10 first mile then 17:50 for 2 miles. That was enough. I don't think that I am caught up on sleep; I should dial back expectations for tomorrow's longish run (maybe 14?)
Math note: I did look up something that I heard about during a talk and thought: "THAT got published?" That gives me more incentive to work on my current paper, which I think is better than some of the stuff I see in print. The editors might not agree though. 🙂
Recovering from a stinging rebuke (resentment and whining alert)
Workout notes: easy 2 mile jog on the treadmill (22:15). I just had breakfast and am waiting for rush hour traffic to lighten up a bit before getting on the road.
Weird FANS note: I did one lap with Centurian John Greene. He is also a math professor; he was working this years event as a volunteer. We..talked math.
He told me about two of his published results. One was about unique factorization domains (UFD): his result showed that every UFD has an "almost Euclidian algorithm". His other was about the trace of 2 x 2 matrices and how the trace behaves under matrix multiplication. It turns out that when 3 matrices are multiplied (in various orders), a certain property is obeyed with probability . Note: the assumptions is that the matrices have real entries and that the entries are selected via a normal probability distribution.
Now how cool is that? I had a moving mathematics seminar. 🙂
Note on yesterday's baseball game and today's hotel stay
Yes, I had club seats (splurge, though I paid a discount price). I was uncomfortable with all of the service (being deferred to by attendants, etc.). I ate breakfast at the hotel. I got the "full" breakfast and ate..what I normally eat. My tummy just can't hold that much anymore. I don't like trying to drive while stuffed.
For me, luxury (middle class luxury anyway) is a big waste of money (even when purchased at discount rates), or at least these luxuries.
I'd never cut it as a Republican. 🙂
Accepting defeat and moving on
This result stung. I won't pretend otherwise. I was struggling 1/3 of the way into the race and that is not a good place to be, at least so early on. I've thought about what went wrong, and it is possibly one of two things:
1. I didn't do enough of the "right kind" of training (e. g. monthly 6-8 hour training sessions) even if my total weekly milage was ok.
2. I just can't do these events any longer.
And yes, there is a bit of envy when I see the successful basking in the glow of their successes …my thinking "dammit, that used to be me". Well, it is not me any longer. When it comes to sports, you are what you do…that is, what you CURRENTLY do, not what you did 10-12-15 years ago.
But there is still plenty of time for redemption. I have some ideas for a math paper (more important than my sports), and I still am on track to attempt to run a marathon this fall, hopefully in less than 5 hours. My "long run pace" is right as is my training mileage, and this weekend's event didn't set me back. So I have goals to work toward, and striving for these, rather than envying others or longing for past successes, is the way to emotionally heal. Nurturing resentments doesn't help.
And there is my home. My wife: yes, I am glad that the fitness bug bit her in a minor way. This should add some quality of life for her and I want to remain encouraging. But: last night, she listened to my whining for about a minute or two and then wanted to know if I remembered her text about what SHE did…parked 2.5 miles away from lunch, walked to lunch, and walked 2.5 miles back. Yeah, I know, my first marathon and 50K were very slow (7:12, 8:40 respectively, but remember I was going at my "I hope to go much longer than this pace") but in my current emotional state, it is hard for me to work up much enthusiasm for my wife's "feat", even if she got a ton of "likes" for it on Facebook, way more "likes" than any of my marathons/50 milers get. 🙂
But that's how it works. A "I got an A on my calculus test" gets more attention than "I published another math paper"; it is WHO you get the kudos from that counts.
My Head Talks
The Dean opens it up (talks about 1:30 or so) and introduces me. Then I talk for about 5 minutes, and then the speaker gives an excellent talk about topology, algebra and the mathematics of financial prediction. So if you haven't met me in person, this is what I sound like.
Creature of habit (and missing workouts)
I have to cover for someone's class, which cuts into my morning workout. Hence today I ran 5 miles over lunch (about 5.2 actually)
Good news: it was very pretty, though there was a breeze. I did 2 lower Bradley loops (1.22-1.24 miles each) in 21:39 (8:44-8:52 mpm) with the laps being 11:05/10:34. I walked the big hill coming out of the park after these two laps, and then slow jogged it home. Hey, 5 miles are better than zero. 🙂
I do have to get new shoes though as my teeth are starting to rattle as I run.
Think of this: suppose you have an ordered list of ingredients of a food stuff (say, water, salt, sugar, gelatin..), the weight of a serving, and the amount of say, protein, fats, sugar (nutritional information on the label).
You can use linear programming (and similar techniques) to determine upper and lower bounds for the various ingredients and have a phone application do this.
Why is this important? Some people have dietary restrictions (e. g. some can't process phenylalanine) and therefore have maximum amounts of this substance they can have in their daily diet.
Who knew?
The walk of shame to the library
Workout notes: 8 mile "run" on the Rivertrail to Glenn Oak Park, up Prospect and into Springdale to the Mausoleum and around that small loop. It was windy and just under freezing; not bad running weather though. The footing was mostly good.
Not shown is the small spur I did on the trail which parallels Perry Ave.; that was iced over so I turned around but I did enough to get 8.
Then I did 10 minutes on the elliptical and a few weightless squats to loosen up.
Note: while out on the "run" I found a dollar bill.
Walk of Shame I took a whole stack of books to the library; they were mostly for a project that didn't work out. But I did check out a new book to look at something else and what did I find in that book?
I put it on social media but that isn't exactly "like-bait" 🙂 But seeing that did remind me that I need to get moving. It has been too long since I've had one of these.
I am thinking about things and I have a mystery that I don't understand…yet. For the mathematically inclined: it has to with speeding up the convergence of an alternating series by using Cesàro summation. For the first two examples I've tried (one conditionally convergent, one absolutely convergent), the convergence sped up by a constant multiplicative factor. That isn't very good for computational purposes, but why "that" factor and not some other one?
- February 2017 (34)
- January 2017 (63)
- December 2016 (32)
- November 2016 (42)
- October 2016 (38)
- September 2016 (44)
- August 2016 (39)
- July 2016 (51)
- June 2016 (48)
- May 2016 (63)
- April 2016 (59)
- March 2016 (75)
- 2008 Election
- 2010
- 2010 election
- 2012 election
- 2014 midterm
- 2016
- Aaron Schock
- Ad
- affirmative action
- Agricultural Commisioner
- aircraft
- Alabama
- alternative energy
- america
- April 1
- arizona
- astronomy
- atheism
- Barack Obama
- barback obama
- Barbara Boxer
- baseball
- basketball
- bicycling
- Biden
- big butts
- bikinis
- bill maher on mosque
- bill richardson
- biology
- blog humor
- Blogroll
- blogs
- blood donation
- Bobby Jindal
- books
- boxing
- brain
- bush-era
- business & economy
- butt
- Cheri Bustos
- civil liberties
- Claire McCaskill
- climate change
- college football
- comedy
- cop
- cosmology
- creationism
- d k hirner
- dark energy
- dave koehler
- deadline
- Democrats
- Dick Durbin
- Dick Morris
- disease
- dk hirner
- draw Mohammad day
- draw Muhammad day
- economics
- economy
- education
- edwards
- energy
- entertainment
- environment
- evolution
- extension
- family
- flu
- football
- Fox News Lies Again
- free speech
- Friends
- frogs
- geese
- glenn beck
- glenn hubbard
- green news
- ground zero mosque
- gwen ifill
- haunting songs
- health
- health care
- Herman Cain
- High Speed Rail
- hiking
- hillary clinton
- history
- hsr
- huckabee
- human sexuality
- humor
- if rich people have to pay taxes
- Illinois
- illness
- immigration. racial profiling
- injury
- internet issues
- interstate highways
- interviews
- Intrade Prediction
- islamophobia
- jan brewer
- jim lehrer
- job
- Joe Biden
- John McCain
- jon stewart
- Judicial nominations
- knee rehabilitation
- lahood
- laughing at myself
- liars
- marathons
- mathematics
- matter
- mccain
- media
- michelle bachmann
- Mid Life Crisis
- Middle East
- Mike Huckabee
- mike's blog round up
- mind
- Mitt Romney
- money
- moron
- morons
- movies
- music
- nanotechnology
- national disgrace
- nature
- Navel Staring
- neuroscience
- newshour
- Newt Gingrich
- north america
- north carolina
- NSFW humor
- obama
- obesity
- Olympic Spandex
- Olympics
- Peoria
- Peoria/local
- Personal Issues
- photos
- physics
- Political Ad
- political humor
- political/social
- politics
- politics/social
- poll
- poor
- poverty
- public policy and discussion from NPR public radio program Science Friday with host Ira Flatow. Science Videos
- pwnd
- quackery
- racewalking
- racism
- ranting
- rebulican party
- recession
- relationships
- religion
- Republican
- republican party
- republican senate minority leader
- republicans
- republicans political/social
- republicans politics
- restaurants
- resume
- rich
- rick perry
- rick santorum
- running
- Rush Limbaugh
- sarah palin
- sb1070
- science
- Science Friday teachers
- Science Friday teens.
- shinkansen
- shoulder rehabilitation
- sickness
- social/political
- space
- spandex
- Spineless Democrats
- sports
- statistics
- stem cells
- stephen colbert
- story
- summer
- superstition
- swimming
- tax cuts
- taxes
- technology
- the colbert report
- Tim Pawlenty
- time trial/ race
- training
- trains
- Transportation
- travel
- ultra
- Uncategorized
- walking
- war on drugs
- wealth
- weight training
- whining
- wise cracks
- workouts
- world events
- yoga
RSSEntries RSS
Comments RSS


text:
Since it was first established, Roman has developed a strong identity as a design led and fashion forward retailer. Our business is fast paced, exciting and aspirational, with our passion and enthusiasm reflected in the brand.
Our stunning collection of every-day wear jumpsuits are both stylish and comfortable for any season.


text:
Sir: I was interested to see the piece about Tissa Balasuriya's excommunication ("How Rome dealt with a turbulent priest", 7 January).
I was with Fr Balasuriya in December at the fourth general assembly of The Ecumenical Association of Third World Theologians (Eatwot) in the Philippines. Fr Balasuriya was a founder member of Eatwot and it was with some shock that 93 participants from 33 countries, mainly in the Third World, heard of his possible excommunication.
It was clear that one of the main reasons was his refusal to sign a profession of faith which included the words: "I firmly accept and hold that the Church has no authority whatsoever to confer priestly ordination on women."
The women members issued a statement that "as women theologians from Asia, Africa, Latin America and the minorities from the US, we deeply appreciate this strong gesture of solidarity with women coming from a male theologian".
The whole conference was later invited to sign a resolution affirming support for Fr Balasuriya and appealing to his superiors and the Pope to give him a fair hearing and stop all proceedings excommunicating him.
Many, like me, involved in the struggle to ordain women in the Church of England have studied Fr Balasuriya's writings and valued his thinking. As a Welsh woman celebrating with my sisters in Wales their ordination to the priesthood this weekend, I find it ironical that while one part of the Church is being more inclusive, another is finding it necessary to censure so harshly an inspiring theologian and priest with a long history of struggling for justice and peace.
Theology Adviser to Christian Aid
Malvern, Worcestershire
- More about:
- Churches
- Theology
- Wales
- Worcestershire


text:
When Raven-Symone was at her heaviest, she wore a girdle to fit into red-carpet styles. Ironically, now that she's slimmed down to a trip 135 lbs – losing nearly 70 pounds – she wears body padding to make her "State of Georgia" character look heavier.
"Georgia started out as a bigger girl, and yes I did wear body pads," she tells The Insider. "I wore the same amount of body pads to make me the size that I was… I had to call the psychiatrist. I'm like 'What's going on?'"
She chatted with the writing team on the show. "I said, 'One, the show shouldn't be about weight,'" she explains. "I said, 'Eventually, I really don't want to wear pads.' As a female, I'm going to fluctuate monthly — please keep that in, I don't care — I'm going to fluctuate monthly, let alone through relationships, let alone, they might add salt more into something that I eat that I don't know about and then I've got to stop eating that."
Raven also set the record straight about rumors that she had a baby in 2009 — and photos online of her alleged child. "One, I'm an African-American. You can tell if you take my weave off," she jokes. "Even if I did date someone of Caucasian descent, my baby wouldn't turn out like that. I think because I was big for so long and at certain times and fluctuated so much that that they thought I had baby weight, just because I was thicker."
While she's open to new relationships, Raven says she's not too worried about finding the right man right away. "When I was 15, I already knew I was going to have three divorces. I already have the prenup written. That's just because I admire Liz Taylor."


text:
News & Updates
Day 1 of the Bendigo NCR Championships and our team put in some fine performances. The weather was a somewhat cool and windy day with periods of warm sunshine that made you hot and then you suddenly you needed a jumper again! For some of our athletes, the Championships were a first time experience which they got a lot out of. Images of the day can be viewed here, or via the Galleries. Good luck to all competing for day 2!
After a very hot start to the first of our club championship rounds, we can look forward to a week off as the Seymour Alternative Farming Expo is on this weekend. However, for those that registered to compete in the NCR Track & Field Championships this weekend over at Bendigo, we wish you the very best of luck and we are sure that you will do your very best to represent yourself, and do the club proud. Good luck to all those attending!!
Local competition resumes the following Friday, 24th February! - See you then!
Welcome everyone, we trust that all our families and athletes had a great Christmas and New Years and are keen to get back into some fun fitness.
Normal competition resumes with Round 11 on Friday 20th January, 2017.
For those athletes in the U/9 to U/15 age groups wishing to compete in the Northern Country Region (NCR) Championships in Bendigo on the 18th & 19th of February, please don't forget to register on the LaVic Website. Please click this link to take you to the registration page. Registrations close Monday 30th of January 2017 at 11:59PM at a cost of around $10 per event.
A maximum of four events can be entered. Good luck to those competing!
Hi Everyone, please be aware that Round 8, Friday December 2nd, 2016 is not cancelled due to a separate event being held (Markets) on the same eve. This should not restrict the clubs activities though parents are reminded to exercise caution that increased traffic around the left hand side of the oval near the netball courts and exhibition hall will be probable. Good luck to everyone competing this Friday night.
Congratulations to our young athletes whom ventured up to Albury for the recent NCR Relay's.
By all accounts they did themselves - and the club proud! - well done guys!
Seymour Little Athletics was well represented at the State Track and Field championships held at Casey Fields, Cranbourne on the 19th and 20th of March, 2016.
The Saturday competition was held in difficult and sometimes cold, windy conditions and track athletes had to push hard through strong head winds on the home straight.
For a small centre such as Seymour, we have a high quality representation at the state and national levels.
Isabella Simonelli represented Victoria recently in Perth, procuring a silver in the U/16 Girls discuss at the Australian Junior Youths Championships held on March 11th - 13th. Isabella also posted a respectable 5th in hammer throw and twelfth in Javelin; an outstanding effort.
Also at the Ausralian Nationals in Perth, Lucy Zotti came a respectable 10th in the in 400m and 11th in the in 800m respectively.
At the State Championships, Lucy secured a third in the 800m and a third in the 400m, capping off a fabulous year of sporting achievement.
Hayden Anderson had a tremendous few days at the championships and won Gold in 70m, Silver in the 200m - with another hard fought Gold in the 100m. An outstanding effort with grit and determination, rewarded in reaching qualifications for nationals. Congratulations Hayden.
Adrianne Timosa has been selected for ALAC and will represent Victoria in the upcoming nationals to be held in Adelaide. All athletes did the club proud and gave it their very best as always.
The following Athletes should be congratulated for their efforts and sportsmanship: Lachlan Aldous, Hayden Anderson, Giselle Andrew, Ben Bath, Steph & Hannah Calderwood, Hannah Cotter, Tom Garner, Angus Garner, Clodagh McCarthy, Isabella Simonelli, Adrianne Tomisa, Ashley Timosa, Tom Wyllie and Lucy Zotti.
Images of the weekend can be found in the Galleries here.
A big thanks to all the parents and family whom assisted in the volunteer work and barracking for our athletes. Special thanks to Scott Calderwood for assisting myself during the meet, which went very smooth. :)
Finally, a big thank you to Kelvin Lubeck for his coaching and support of the athletes throughout the year. With his help, many athletes have benefited from his wisdom and knowledge and progressed their personal bests and awards.
Wayne Aldous & Scott Calderwood.
Team Managers for the day.
Seymour Little Athletics Centre (SLAC36) is a member of the Little Athletics Victoria (LAVic), Northern Country Region. The Northern Country Region is one of seven regions in Victoria and consists of the following clubs and centres: Albury, Barooga, Benalla, Bendigo, Cowora, Echua/Moama, Howlong, Kyabram, Kyneton, Lavinton/Jindera, Mansfield, Murrandindi, Mt. Beauty, Nathalia, Rochester, Rutherglen, Seymour, Shepparton, Wangaratta, Wodonga and Yarrawonga/Malawa.
Seymour Little Athletics started in 1968 and became the 36th Centre to form in Victoria. In the early 1970's, Seymour held Open Days and a gift, which was held at Kings Park.
The inter club competition at Seymour takes place at Kings Park on Friday nights, commencing the 14th of October 2016, and concludes in March 2017. Competition starts at 5.30pm and runs for approximately 2 hours.
Training nights are held at Kings Park, Seymour, each Monday night, starting in October 2016 - this time is the perfect opportunity for competitors to hone skills in specific disciplines.
Training starts at 5:00 pm, and usually goes for an hour, subject to demand.


text:
Korg Monotron Duo Analog Ribbon Synthesizer
- Write a Review
Korg's monotron DUO is a ribbon-controller keyboard that gives you not just one, but two analog oscillators to create an even more extreme sound. If ...Click To Read More About This Product
6-Month* financing available.
- Free Shipping
- Top Rated
- Top Seller
- Lowest Price
- 45 Day Returns
Palm-sized ribbon controller keyboard featuring two analog oscillators and VCF filter and more tweaking fun.
Korg's monotron DUO is a ribbon-controller keyboard that gives you not just one, but two analog oscillators to create an even more extreme sound. If you tune the oscillators in unison, you get a rich, thick, analog tone. If you tune them in intervals, you can emulate classic soloing sounds and generate new effects. Tweak the X-MOD (cross modulation) circuit taken from Korg's classic Mono/Poly and you can introduce a mild or razor-sharp edge to the overall sound; everything from a rich vibrato to extreme metallic sounds reminiscent of an FM synth.
VCO + VCF = Modular synthesis building blocks
The basic analog sound-building circuit in the monotron DUO is radically simple: one voltage-controlled oscillator and one voltage-controlled filter. As a result, the process of sound creation is intuitive, and instantly understandable.
Enhanced Ribbon Controller Keyboard
The monotron DUO's ribbon controller keyboard now features the familiar Scale function used on the KAOSSILATOR. You can use this button to select one of four scales: Chromatic, Major, Minor, or Off. Choose the Chromatic scale to play precise pitches as on a piano, or choose Major or Minor to generate musically coherent phrases by simply sliding your finger around on the keyboard.
Go Anywhere Analog
A miniature speaker is built into the monotron DUO. Along with the battery operation and compact size, this feature allows you to enjoy the share the monotron DUO's analog synthesis sound with your friends, anywhere, at any time. Use it to entertain friends, for recording, or even at karaoke during a musical interlude for solos. It also has a headphone output you can use when practicing in private, creating sounds at home, recording, or for connecting to an amplifier or PA system.
Classic Korg Filtering
The distinctive tone of an analog filter (VCF) can dramatically alter and enrich the sound. The monotron DUO uses the same highly acclaimed VCF circuit as KORG's classic MS-10 and MS-20 semi-modular synthesizers. Originally released over 30 years ago, these instruments are still coveted today for their aggressive, dynamic filters. The VCF Cutoff knob sets the frequency or tone of the filter; the VCF Peak knob adds emphasis. A powerful filter is an essential aspect of any analog synthesizer, applying dramatic tonal changes to the sound, and projecting an intense personality that will stand out, especially in any live performance.
Fully-patchable Filter
The input jack (AUX input) built into the monotron DUO's compact body allows you to place any audio source into the signal path, directly before the analog filter. This provides a great way to connect a KAOSSILATOR, a guitar, or a digital audio player to the monotron DUO and enjoy tone-bending filter effects.
- Two analog oscillators (VCO) with individual tunings
- Edgy X-MOD (with Intensity knob) circuit from Korg's classic Mono/Poly
- VCF with Cutoff and Peak Control
- Ribbon controller keyboard with Scale Select
- Original Analog (VCF) Filter taken from the classic MS-10 & MS-20
- Aux input jack lets you apply filter to any audio source
- Built-in speaker and battery power for Go Anywhere Analog sound
Get lots of fun at your fingertips with the monotron DUO! Order now!
Monotron Duo Analog Ribbon Synthesizer
- Synthesizer structure: 2 VCOs (Square Wave) with X-Mod; 1 VCF
Switches: Standby / VCO1 / VCO1+2
Scale Select Button: (Chromatic -> Major -> Minor -> Off)
Knobs: VCO1 Pitch, X-MOD Int., VCO2 Pitch, VCF Cutoff, VCF Peak
Controller: Ribbon Controller with Scale SelectVolume: Headphone Volume
Aux Input: 1/8" stereo jack
Headphone Output: 1/8" stereo jack
Speaker: Miniature internal which is disabled when headphones are in use
Power Supply: 2 AAA batteries included, recommended alkaline batteries
Battery Life: Approximately 8 hours (with alkaline batteries)
Dimensions (WxDxH): 120mm x 72mm x 28mm / 4.72" x 2.83" x 1.10"
Weight: 95g / 3.35 oz. (without batteries)
Reviewed by 1 customer
- Easy to Use
- Great sound tweaking
- Portable
- Realistic Sound
- Responsive / Good Action
- Touch Sensitive Keys
- Poor speakers
Best Uses
- Accompaniment
- Electronic Music
- Learning
- Travel
Comments about Korg Monotron Duo Analog Ribbon Synthesizer:
When I opened the box that had the Duo synth in it I was not expecting for how small of a device it was. Nor how big the sound was soon as I plugged in my headphones.
With my big fingers its hard to use the ribbon, but I am adjusting as it is sensitive.
I had alot of fun with the aux input and it was pretty cool getting to use the VCF to mess with the sound coming in and play along with the music.
I especially got it because I need an instrument while I am in training since there is not enough room for my larger instruments in the barracks.


text: Manhattan in the Desert may be outside of your area. Please enter your delivery address to check availability Do you need any... Napkins & Utensils Continue to Checkout Manhattan in the Desert Back to Menu Your order refresh Manhattan in the Desert Your Shopping Cart is empty edit Delivery Address Please enter delivery address edit Delivery Time N/A* Add someone to this order Ravioli Meat or Cheese ravioli topped with Meat Sauce or Marinara and mozzarella cheese. What Kind Of Ravioli?Select one of the choices Prepare Meat Ravioli Prepare Cheese RavioliPrepared With Marinara Or Meat Sauce?Select one of the choices Prepare With Marinara Sauce Prepare With Meat SauceAdd Side Salad?You may select one of the choices Add Side Caesar Salad; Caesar Dressing On The Side ($2.99) Add Side Garden Salad; Ranch Dressing On The Side ($2.99) x $10.99 Special Instructions: Label Item For: or Cancel


text:
Explore nine marked natural trails and discover the boreal forest, the taiga and the tundra. As you walk across one of North America's oldest volcanoes, Mont Laurier, you'll observe nature's victory over this rugged climate, mainly in the form of rare lichens and mushrooms that are thousands of years old.
Telephone : 819 739-4566E-mail
Bureau d'information touristique de Matagami, 100, place du Commerce, Matagami, Québec, Canada, J0Y 2A0
Activities and services
- Nature interpretation
- Hiking : 30 km
- Snowshoeing : 30 km
- Pets allowed
- Documentation / publications
- Camping accommodation
- Shelter-accommodation : 1
- Panoramic view / lookout
Vehicle and Parking Services
- Free outdoor parking
Rental services
- Snowshoes