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General features of Schizosaccharomyces pombe.
Schizosaccharomyces pombe (Sz. pombe), or fission yeast, is an ascomycete unicellular fungus that has been used as a model system for studying diverse biological processes of higher eukaryotic cells, such as the cell cycle and the maintenance of cell shape, apoptosis, and ageing. Sz. pombe is a rod-shaped cell that grows by apical extension; it divides along the long axis by medial fission and septation. The fission yeast has a doubling time of 2–4 hours, it is easy and inexpensive to grow in simple culture conditions, and can be maintained in the haploid or the diploid state. Sz. pombe can be genetically manipulated using methods such as mutagenesis or gene disruption by homologous recombination. Fission yeast was defined as a micro-mammal because it shares many molecular, genetic, and biochemical features with cells of higher eukaryotes in mRNA splicing, post-translational modifications as N-glycosylation protein, cell-cycle regulation, nutrient-sensing pathways as the target of rapamycin (TOR) network, cAMP-PKA pathway, and autophagy. This chapter uses Sz. pombe as a useful model for studying important cellular processes that support life such as autophagy, apoptosis, and the ageing process. Therefore, the molecular analysis of these processes in fission yeast has the potential to generate new knowledge that could be applied to higher eukaryotes.
- Yeast
- Schizosaccharomyces pombe
- cellular model
- autophagy
- apoptosis
- ageing
- chronological lifespan
1. Introduction
1.1. General features of Schizosaccharomyces pombe
Schizosaccharomyces pombe (Sz. pombe) is a yeast found in sugar-containing fermentations of alcohol involved in the production of beer in subtropical regions; the yeast was first described in 1893 by P. Lindner who isolated yeast from millet beer of East Africa and gave it the name "pombe" from the Swahili word for beer. Sz. pombe is a free-living fungus, classified with ascomyctes such as S. cerevisiae (Table 1). However, fission yeast is taxonomically and as evolutionarily distinct from budding yeasts as it is from humans. Sz. pombe reproduces by fission, a process similar to that used by higher eukaryotic cells. Despite the variation in the predicted time of divergence, phylogenetic analysis, and experimental evidence, it can be shown that the gene sequences of Sz. pombe are often more similar to its counterpart in mammals than the equivalent of genes of S. cerevisiae [1].
Sz. pombe is described as a fission yeast because it grows by apical extension and it divides along the long axis by medial fission and septation. The fission yeast has a doubling time of 2–4 hours, and can be maintained in the haploid or the diploid state. The yeast has a cylindrical rod-shape 3–4 μm in diameter and 7–14 μm in length [2]. The cell wall of Sz. pombe provides mechanical strength and protection from environmental stresses. The cell wall determines cellular morphology during the different stages of the life cycle, and is continuously remodelled during the cell cycle to allow cellular growth. The cell wall of Sz. pombe is composed of 74–82% glucan and 9–14% galactomannan, and has a chemical composition of β-1, 3-glucan, β-1, 6-glucan, α-1, 3-glucan, and α-galactomannan. Viewed by electron microscopy, the cell wall is organized in a three-layered structure: the outer layer is electron-dense, the adjacent layer is less dense, and the third layer bordering the cell membrane is dense [4, 5].
1.2. Genome organization of Sz. pombe
The whole genome of Sz. pombe, distributed over three chromosomes, is estimated at 13.8 Mb [6]. The genome of Sz. pombe was fully sequenced using 452 cosmids, 22 plasmids, 15 BAC clones, and 13 PCR products, in a project under the control of Schizosaccharomyces f European Sequencing Consortium (EUPOM), the Wellcome Trust Sanger Institute (United Kingdom), and Cold Spring Harbor Laboratory (USA). The collaborating bodies were Funding Agency Cancer Research UK (Cambridge Institute United Kingdom) and the European Union (EU) [7]. The genome is organized in the chromosomes I (5.7 Mb), II (4.6 Mb), and III (3.5 Mb), and a 20-kb mitochondrial genome. The three centromeres are 35, 65, and 110 kb long for chromosomes I, II, and III, respectively. On the centromeres are 174 tRNAs genes, 45 of which have introns. The genome of the yeast has at least 4, 940 protein-coding genes (including 11 mitochondrial genes) and 33 pseudogenes. Sz. pombe introns average only 81 nucleotides in length. The gene density for the complete genome is one gene every 2, 528 bp, which is similar in chromosomes II and I (one gene every 2, 483 and 2, 457 bp, respectively) and is less dense for chromosome III (one gene every 2, 790 bp). The spliceosomal RNAs (U1±U6) are located with 16 small nuclear RNA genes (snRNAs) and 33 small nucleolar RNAs (sno-RNAs). The 5.8S, 18S, and 26S ribosomal RNA genes are grouped as 100±120 tandem repeats in two arrays on chromosome III, with the 5S ribosomal RNA genes being distributed throughout the genome [7]. In silico analysis of the Sz. pombe genome has made it known that there are genes highly conserved between the vegetable and animal kingdoms, some of which have been implicated in cancers in humans [8, 9].
2. Mating-type locus, heterothallic, and homothallic phenotype
Sz. pombe has been studied extensively since the 1950s. Urs Leupold isolated the Sz. pombe strains widely used in research from a culture of Sz. pombe Lindner str. liquefaciens obtained by Osterwalder [10] from grape juice in France, in 1921. In its normal life cycle, Sz. pombe cells are haploids, although they can grow in a diploid state. Leupold described the haploid character and the complexity of the mating-type genetics of Sz. pombe, by culturing the haploid and diploid yeast [11, 12, 13], which were both homothallic and heterothallic (h) strains. The mating of Sz. pombe works by controlling mat genes located on the mating-type locus in the chromosome II [14], which is organized by one expression locus (mat1) and two donor loci mat2 and mat3 that encode the mat P and mat M genes, respectively. There are three alleles mat for the mating-type: h+ (strain 975) or P (Plus, +), h− (strain 972) or M (Minus, -), and h90 (strain 968). The mating-type is determined by the mat1 locus, cells that present the segment called mat1-P determine the sexual type P or h+ via the two genes mat-Pi and mat-Pc [15]. Cells with the segment mat1-M define the sexual type M or h− by two genes mat-Mi and mat-Mc. The P and M alleles determine the mating reactions of the heterothallic strain. The homothallic strains with cells of both the P and M mating type can mate and form asci with four ascospores.
The heterothallic strain with only one mating type, are able to mate with heterothallic Sz. pombe strains of the opposite mating type. The allele h90 determine the homothallism [11], derived from P and M by recombination and mutation by the crossing of heterothallic strains. Homologous sequences flank the mating-type locus support site-specific recombination between the expression locus and the donor loci (mat1 and mat2, or mat1 and mat3) [16]. Heterothallic strains in Sz. pombe originated from strains having spontaneously rearranged their mating type loci. For homothallic segregants with the markers h+ parent and recombination for two marker genes (his7 and his2) flanking the locus, two mating-type genes are generated and named initially as h1 and h2 and later renamed mat1 and mat2, respectively [17].
|Doubling time of 2–4 hours
Cylindrical rod-shaped
3–4 μm in diameter and 7–14 μm in length
Ascospores arranged linearly
Easy genetic manipulation and mutant generation
|NCBI Taxonomy ID: 284812
Schizosaccharomyces pombe
|Percentage in the full cell cycle
|S (Cytokinesis)
|Fission yeast can exist stably in either haploid or diploid states.
|Heterothallic P
|Heterothallic M
|Gene density
|Coding (%)
|Length (Mb)
|Whole genome
|Centromere I 35 kb
|Centromere II 65 kb
|Centromere III 110 kb
|Mitochondrial genome
|20 kb
|Count of known and putative peptidases121
non-peptidase homologues 39
3. Cell cycle of Sz. pombe
Sz. pombe coordinate their growth with the cell cycle. Exponentially growing cells are of a similar cell size and double in mass before entering the next round of cell division. Cell length is a measure of the stage of the cell cycle that the cell has reached [18]. The cells are commonly cylindrical with rounded ends, but under conditions of starvation cells become shorter. Sz. pombe is a single-celled, haploid organism with a cell cycle typical of a eukaryote, organized in the same stages as those of higher eukaryotes: interphase (G1, S, and G2 phases) and mitosis by mechanisms that are similar to those in multicellular animals. Sz. pombe usually proliferates in a haploid state. In a medium rich in nutrients, wild-type cells undergo a mitotic division approximately every two hours, with a short G1 phase taking less than 10% of the cell cycle. Next, the cells enters into the S phase following cytokinesis; the longer stage of the cell cycle is the G2 phase (70% of the cell cycle) [19]. Cells sense their size and use this information to coordinate cell division with cell growth to maintain a constant cell size by Pom1 protein and Nif1, localized to the cell ends of DNA, and inhibits mitotic activators in the middle of the cell acting as sensors of cell size controlling the onset of mitosis [20]. When starved of nitrogen, cells stop the division and arrest the cycle in G1, which promotes the sexual phase of the life cycle through conjugation of pairs of P and M cells to form diploid zygotes in a process known as karyogamy. In nitrogen-starved conditions, Sz. pombe haploid cells expressing the mat P and mat M genes enters into meiotic division after forming zygotes [21, 22]. After mating and nuclear fusion, premeiotic replication occurs, DNA content is duplicated from 2N to 4N, there is pairing and recombination of homologous chromosomes, and ocurrs the reductional meiosis I and the meiosis II division for the production of four haploid nucleic (1N), which are separated into four spores in an ascus, where the ascospores of Sz. pombe are arranged linearly [13]. Sz. pombe is normally haploid, but it is possible to select diploid strains, which can then divide by vegetative mitotic growth until starved of nitrogen, when they undergo meiosis and form azygotic asci. The provision of a rich nutrient source induces new vegetative growth and mitotic cell division. In 1957, Mitchison measured changes in the volume of cells along the yeast cell cycle. He found that volume increased slightly in the interphase, then changed about an hour before the cell split and increased just after cleavage. Sz. pombe extended at one end only, usually the older one [23, 24]. The cell cycle of Sz. pombe is described in Figure 1.
4. Cellular process studied using Sz. pombe
Sz. pombe is an excellent model for the study of common eukaryotic cellular processes. The yeast shares important biological aspects with metazoans, such as chromosomal structure and metabolism, relatively large chromosomes and centromeres, low-complexity replication origins, epigenetic mechanisms for regulation of genetic expression, G2/M cell cycle control, cytokinesis, mitosis and meiosis, DNA repair and recombination, the mitochondrial translation code, and spliceosome components, among others [2, 25].
Fission yeast presents a short cell cycle and is easy to manipulate by genetic classical and molecular analysis. Sz. pombe is a particularly useful model for the study of the function and regulation of genes from more complex species [26]. A model organism is any species used for scientific research in order to answer a specific question that cannot be studied in any other way. A model must be amenable to experimental manipulation in the laboratory, require low costs of culture and maintenance, have a short generation time, be amenable to genetic manipulation to produce mutants and allow the study of their effects, and show high conservation of mechanisms or specific cellular process. A system model extensively used in microbiology, biochemistry, biotechnology, and molecular biology is Escherichia coli (E. coli). Although this bacterium can be grown and be cultured easily and inexpensively in a laboratory setting, E. coli lacks the mechanisms for post-transcriptional on mRNA or post-translational modifications on proteins of eukaryotic origin. Saccharomyces cerevisiae (S. cerevisiae) is other model used due to its quick, inexpensive, and easy growth in control conditions of the laboratory. However, like E. coli, S. cerevisiae has a limited capability for performing post-transcriptional or post-translational modifications such as N-glycosylations. Drosophila melanogaster is other model system in the cellular biology field widely used for classical genetics analysis. Caenorhabditis elegans is the model of choice for understanding the genetic control of development, physiology, and the study of cellular process such as apoptosis and ageing. Mus musculus and Rattus norvegicus are two mammalian models used for the study of several diseases that affect humans; produced by highly conserved genes, its main disadvantage is slower reproduction than other models allow. In contrast, fission yeast is an ideal system for the analysis of cellular processes common in eukaryotic cells; since it shares many molecular, genetic, and biochemical characteristics with multicellular organisms — such as the splicing of mRNA, post-translational modifications, cell division, and cell cycle control [13, 27, 28] — it has a high rate of reproduction in a short period.
4.1. Model for studying the eukaryotic cell cycle
Sz. pombe, the fission yeast, has been used as a model for the study of the eukaryote's cell cycle. Fission yeast is a powerful tool for the study of the cell cycle, since it has a typical cycle cell eucarionte G1, S, G2, and M. G2 occupies approximately 70% and the remaining stages on 30% of the total duration of the cell cycle [29, 30]. Since it has a typical cell shape, it is a useful system for identifying genes involved in the cell cycle because mutants are easily identifiable when compared with the wild type [31]. In the 1970s, using Sz. pombe P. Nurse demonstrated the cyclin/cyclin-dependent kinase complex as the key regulator of cellular cycle. Sz. pombe has a single mitotic cyclin-dependent kinase (CDK), highly conserved, named cdc2p, homologous to CDC28 from S. cerevisiae and CDK1 from human cells [32]. Sz. pombe has been used for the molecular sexual differentiation analysis. In this system, it was discovered that a cluster of genes associated with the main biological events of the differentiation process are up-regulate: genes induced in response to environmental changes (starvation and pheromone-induced genes), early genes (pre-meiotic S phase and recombination), middle genes (meiotic divisions and early steps of spore formation), and late genes (spore maturation) [33]. In addition, the use of the fission yeast has contributed to the knowledge of other areas such as cytokinesis, the formation of microtubules, differentiation, cell morphogenesis and polarity, mechanisms of response to stress, and response to damage of the DNA [34, 35].
4.2. Cellular ageing
The identification of evolutionarily conserved mechanisms that determine long lifespans has been of great interest, making the fission yeast Sz. pombe a novel model for the study of ageing. Ageing is a genetically programmed event [36, 37] and can be defined as a decline in biological functions due to the accumulation of molecular and cellular damage imposed by intrinsic and extrinsic factors on living organisms, having mortality as a consequence. Lifespans of humans are measured chronologically from birth to death. Ageing can considered from the perspectives of two types of cellular lifespans: the replicative lifespans (RLS) of continuously dividing cells and the chronological lifespans (CLS) of post-mitotic cells, from terminally differentiated cells. RLS is the number of mitotic divisions a cell can undergo before senescence, and CLS is the length of time a non-dividing cell can remain viable. CLS measures how long cells can remain viable in a non-dividing state, allowing a continuous decline in viability without detectable re-growth until all cells in the culture are dead. The complexity of human bodies and the length of the human lifespan make research on the biology of human ageing difficult. Model organisms such as yeast, worms, and flies have been extensively utilized because of their short lifespans and their accessibility to classical genetics techniques, as well as the high degree of conservation of genes, processes, and signalling pathways that they share with humans and other mammals [38]. Some aspects of ageing studied in fission yeast have been mainly focused on the effect of calorie restriction on ageing and on the detection of nutrient signalling pathways [39, 40]. The protein kinase target of rapamycin (TOR) and insulin/insulin-like growth factor (IGF)-1 pathway are the well-studied nutrient sensing pathways that determine the length of lifespan [41]. TOR exerts its function through two distinct complexes: TOR complex 1 (TORC1) and TOR complex 2 (TORC2), which regulate translation, cell growth, and the organization of actin cytoskeleton [42, 43]. Inactivation of TORC1 increases the lifespan in model organisms such as budding yeast, worms, flies, and mice [44, 45]. The insulin/IGF-1 signalling pathway, and their components such as PI3 kinase and AKT kinase, affect the longevity of flies, worms, and mice [39]; interestingly, this pathway has also been found in yeasts with the AKT kinase. In S. cerevisiae, nutrient signalling via the kinases Sch9/Tor/PKA pathway has a strong pro-ageing effect. Down-regulation of this pathway is partially responsible for CLS increase. In fission yeast, there are two homologues of these kinases with a similar pro-ageing activity in Sz. pombe: pka1 y sck2 [46]. In this model, deletion of pka1 or sck2 extended CLS by diminishing the accumulation of ROS and increasing oxidative stress resistance [47, 48]. AKT kinase orthologues of Sz. pombe regulate CLS: loss of sck1 extended the lifespan in the condition with the excess of nutrients and loss of both genes showed that sck1 and sck2 control different longevity pathways. In addition, there are four genes identified as regulators of ageing in Sz. pombe: 1. psp1+/sds23+/moc1+, a gene required for long-term survival in the stationary phase. Psp1 is phosphorylated in the stationary phase by the cyclin-dependent kinase complex Cdc2/Cdc13. 2. Casein kinase cka1+, an orthologue of the α-subunit of casein kinase II in mammals, whose function is essential but the details of the effect of its deletion are not known. 3. adh1+, encoding the alcohol dehydrogenase that converts acetaldehyde to ethanol. 4. rpb10+, a protein subunit shared by RNA polymerases I, II, and III. It is noteworthy that lifespan extension from TOR-inhibition, Sch9/Akt, or caloric restriction is an effect highly conserved between yeast, worms, and flies [39]. Research of ageing on fission yeast has greatly benefited the knowledge of molecular process since the data obtained can be extrapolated to mammalian cells.
4.3. Autophagy
Autophagy is a catabolic mechanism that regulates the intracellular turnover of unfolded/misfolded, long-lived, or damaged proteins, lipids, and organelles (such as mitochondria and the endoplasmic reticulum (ER), through its sequestration within a double-membrane and delivery to lysosomes for degradation and recycling of biocomponents. Under basal conditions, autophagy is a housekeeping programme, but it can also be activated by nutrient starvation, low cellular energy levels, amino acid deprivation, growth factor withdrawal, ER stress, hypoxia, oxidative stress, and infection [50–54]. Autophagy deregulation in higher eukaryotes leads to muscle atrophy, myopathy, and cardiac and immune disease [52–54], and plays a dual role in the pathogenesis of cancer [50, 52, 55, 56].
Duve [57] introduced the term autophagy from the Greek auto (itself) and phagy (eat) which means that cells degrade their cytoplasmic components through lysosomal degradation. Clark described the autophagy process in 1957 through electron microscopy of the kidneys of newborn mice [58]; he discovered that inside lysosomes there were mitochondria. Unfortunately, it was very difficult to study autophagy via electron microscopy and biochemical analysis; thanks to the introduction of molecular techniques, it was later discovered that the yeast S. cerevisiae was a tractable model of eukaryotes. Thirty Atg proteins (autophagy-related proteins) were also discovered later, and it was found that they possess mammalian orthologues [50–52, 54, 59–61]. Four types of autophagy have been described in mammals (Figure 2): macroautophagy (hereafter referred to as autophagy), microautophagy, chaperone-mediated autophagy (CMA), and the more recently described RNautophagy [61–64]. In autophagy, the cytoplasmic components (also known as cargo) are first encapsulated in a double-membrane structure synthesized de novo, which at the beginning is the phagophore and when it is completely formed is called autophagosome (AP), after which it is trafficked and fused to the lysosome. In microautophagy the cargo is selected through an invagination in the lysosome where it is degraded. In the CMA, specific cytosolic proteins containing the motif KFERQ are recognized by the heat shock cognate protein 70 (HSC70), and are then delivered to the lysosomal associated membrane protein (LAMP2) of type A receptor that is located in the lysosome membrane, where the proteins are translocated inside and degraded [62–64]. In 2013, Fijiwara [65] described RNautophagy, where several RNAs were recognized by the LAMP2C receptor and degraded in the lysosome. However, autophagy is a very complex process in metazoans, making it important to have simplest biomodels that share with mammals the core of the autophagic machinery, such as the budding yeast S. cerevisiae and the fission yeast. In the budding yeast autophagy, microautophagy and cytoplasm to vacuole targeting (CVT) have been outlined; likewise in the fission yeast, autophagy shares almost the same conserved core of autophagic machinery (Figure 2). However, microautophagy, CVT, and RNautophagy have not been described and further investigation is needed in order to unravel the conservation of these mechanisms.
The Atg machinery was classified into five groups according to function in each step of the autophagic process (Table 2 and Figure 3) [1–6, 11, 12, 50–55, 60, 63, 64]: ULK1 kinase and its regulators that signal the autophagosome biogenesis, the phosphatidylinositol (PtdIns) 3- kinase complex controls the nucleation step that recruitsother Atg proteins hierarchically, the elongation of the phagophore mediated by the Atg12 and LC3 conjugation systems, and a subgroup of proteins with unknown functions. In mammals under nutrient rich conditions, insulin or growth factors activate the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. When PI3K is activated, it converts phosphatidylinositol (3, 4)-bis phosphate (PIP2) to phosphatidylinositol-3 phosphate (PIP3), then Akt is activated and inactivated by phosphorylation to TSC which leads to mTOR complex 1 (mTORC1) activation. mTORC1 negatively regulates ULK1 and Atg13, so when mTORC1 is inactivated by nutrient depletion, low concentration of insulin, deprivation of amino acids, or the addition of its antagonist rapamycin, the ULK1 kinase and Atg13 are then activated, forming the ULK1 kinase complex (Table 2), which signals the induction of the AP formation. There is another inductor of autophagy, the AMP activated protein kinase (AMPK), that senses low intracellular energy status and directly activates by phosphorylation to ULK1, and by inhibiting mTORC1 via phosphorylation of Raptor (a subunit of mTORC1) [66–69]. ULK1 phosphorylates Beclin1 at serine 14, which is a component of the PtdIns 3- kinase complex (Table 2) and enhances Vps34 activity — this step is crucial in the AP formation because Vps34 inhibition by 3-methyladenine (3-MA) or wortmannin disrupts the biogenesis. Vps34 synthesizes PIP3 at the sites where AP are assembled; it has been suggested that PIP3 recruits additional factors for AP formation, but its role remains unclear. Beclin1 binding proteins that activate or inhibit AP biogenesis have been identified: the UV radiation resistance-associated gene protein (UVRAG) and Bcl-2 or Bcl-XL, respectively. Beclin-UVRAG interacts with the class C Vps complex proteins, which are part of the endosomal fusion machinery, so this interaction induces AP fusion with lysosomes [50, 53, 70–71].
The next step in AP building is the elongation of the phagophore, which requires membrane input from organelles (such as ER, mitochondria, cytoplasmic membrane, or possibly from de novo synthesis); however, the exact membrane source is unknown. In mammals, Atg9 is a transmembrane protein that probably carries membrane expansion of the phagophore but its downstream effectors have not yet been elucidated. However, there are two ubiquitin-like complexes that are required for elongation; the first one is the Atg12 conjugation system (Table 2), in which the ubiquitin-like protein Atg12 is covalently joined to Atg5. This must first be activated by Atg7 (E1 ubiquitin activating enzyme-like or E1). Atg7 uncovers Atg12 glycine carboxyl-terminus, then Atg12 is transferred to Atg10 (E2 ubiquitin conjugation enzyme-like or E2), after which it is covalently attached by its terminal glycine to lysine 130 (Lys 130) of Atg5.
Finally, Atg12-Atg5 conjugates with Atg16L1 forming a tetramer; this tetramer is essential for the elongation but when AP has been completed it dissociates [76–78]. The second ubiquitin-like complex is the microtubule-associated protein 1 light chain 3 (LC3) conjugation system (Table 2). LC3 is a precursor of LC3-I, it is obtained by the cleavage of the protease Atg4B to its carboxyl terminus. LC3-I is attached to phosphatidylethanolamine (PE) by the enzymes E1 and E2- ubiquitin-like Atg7 and Atg3, respectively, obtaining the LC3-II product which forms part of the AP. Notice that the LC3 complex lacks an E3 ubiquitin ligase-like enzyme that could facilitate LC3I-PE conjugation; however, a crosstalk between these two ubiquitin-like systems has been demonstrated by the tetramer Atg12-Atg5-Atg16L1 acting as E3-like enzyme. When AP is formed, LC3 can be recycled from the form LC3II; this is achieved through Atg4 which breaks apart LC3I from PE, and LC3-I and LC3-II assays are widely used for monitoring autophagy [60, 77]. Finally AP moves bidirectionally along microtubules towards the microtubule organizing centre (MTOC) where lysosomes are enriched, then AP fuses with lysosomes forming autolysosomes where the content is degraded and the components are recycled [50]. Atg2 is required for the AP location and can possess a different role when binding to Atg18 in order to target the complex to autophagic membranes [79]. In Sz. pombe, depletion of nitrogen from the culture medium triggers autophagy. Unlike mammals, the addition of AMPK does not induce autophagy, nor can rapamycin induce it. It has been reported that rapamycin has a partially inhibitory effect on TOR activity, moreover, the addition of rapamycin plus caffeine prompts a starvation-specific gene expression pattern and autophagy [80–81]. Atg 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 18a, 18b, 18c, Vps34 homologues have been found (Table 2) [73–75], and their deletion inhibits AP formation.
% identity
|Sz. pombe
(% identity)
|ULK1 kinase complex
M. musculus: 89%
R. norvergicus: 89%
|Kinase ser/thr induces autophagy and PA assembly [72, 73–75]
M. musculus: 94%
R. norvergicus: 89%
|Regulator and binding protein of ULK1 complex [72, 73–75]
M. musculus: 89%
R. norvergicus: 89%
|Binding protein and regulator of ULK1, induces PA biogenesis [72, 73–75]
M. musculus: 99%
R. norvergicus: 98%
|Binds to Atg13 in the ULK-Atg13-FIP200 complex, which is important for the stability and basal phosphorylation of Atg13 and ULK1 [72, 73–75]
|PtdIns 3- kinase complex
M. musculus: 96%
R. norvergicus: 96%
|Regulatory kinase subunit of the PtdIns 3- kinase complex [72, 73-75]
M. musculus: 93%
R. norvergicus: 98%
|Necessary for the function of the PtdIn2-kinase complex [72, 73–75]
M. musculus: 97%
R. norvergicus: 98%
|PtdIns 3- kinase produces PI3P and allows the recruitment of PI3P binding proteins like WIPI1/2 and the two conjugation systems [72, 73–75]
M. musculus: 98%
R. norvergicus: 97%
|Modulates AP biogenesis by binding to Vps34 [72, 73–75]
M. musculus: 88%
R. norvergicus: 94%
|Binds Beclin1 enhancing AP biogenesis [72, 73–75]
|Atg12 conjugation system
M. musculus: 76%
R. norvergicus: 71%
|Ubiquitin-like protein covalently joined to Atg5 [72, 73–75]
M. musculus: 97%
R. norvergicus: 95%
|Ubiquitin-like protein covalently attached to Atg12 and interacts with Atg16 [72, 73–75]
M. musculus: 89%
R. norvergicus: 89%
|E1-like enzyme for Atg12-Atg5 and LC3-PE formation [72, 73–75]
M. musculus: 72%
R. norvergicus: 79%
|E2-like enzyme required for Atg12-Atg5 complex
In Sz. pombe, SpAtg10is also required for normal cell cycle progression and for responses to several stress agents but has no role in autophagy [72, 73–76]
M. musculus: 82%
R. norvergicus: 82%
|Necessary for the assembly of the tetramer Atg12-Atg5-Atg16 [72, 73–75]
|LC3 conjugation system
M. musculus: 83%
R. norvergicus: 83%
|Conjugated with PE forms part of the AP membrane [72, 73–75] [22, 23–25]
M. musculus: 91%
R. norvergicus: 93%
|E2-like enzyme [72, 73–75] [22, 23–25]
M. musculus: 92%
R. norvergicus: 91%
|Induces LC3-II then helps to recycle LC3 [72, 73–75]
M. musculus: 89%
R. norvergicus: 98%
|E1- like enzyme [72, 73–75]
|Atg2-Atg18 complex and Atg9
M. musculus: 85%
R. norvergicus: 84%
|Required for AP localization [72, 73–75]
|Required for AP localization [72, 73–75]
M. musculus: 94%
R. norvergicus: 94%
|In Sz. pombe is a protein required for the targeting of Atg12-Atg5-Atg16 complex [72, 73–75]
M. musculus: 98%
R. norvergicus: 98%
|Transmembrane protein that might carry membrane expansion of phagophore [72, 73–75]
Finally, Atg12-Atg5 conjugates with Atg16L1 forming a tetramer; this tetramer is essential for the elongation but when AP has been completed it dissociates [76–78]. The second ubiquitin-like complex is the microtubule-associated protein 1 light chain 3 (LC3) conjugation system (Table 2). LC3 is a precursor of LC3-I, it is obtained by the cleavage of the protease Atg4B to its carboxyl terminus. LC3-I is attached to phosphatidylethanolamine (PE) by the enzymes E1 and E2- ubiquitin-like Atg7 and Atg3, respectively, obtaining the LC3-II product which forms part of the AP. Notice that the LC3 complex lacks an E3 ubiquitin ligase-like enzyme that could facilitate LC3I-PE conjugation; however, a crosstalk between these two ubiquitin-like systems has been demonstrated by the tetramer Atg12-Atg5-Atg16L1 acting as E3-like enzyme. When AP is formed, LC3 can be recycled from the form LC3II; this is achieved through Atg4 which breaks apart LC3I from PE, and LC3-I and LC3-II assays are widely used for monitoring autophagy [60, 77].
AP moves bidirectionally along microtubules towards the microtubule organizing centre (MTOC) where lysosomes are enriched, then AP fuses with lysosomes forming autolysosomes where the content is degraded and the components are recycled [50]. Atg2 is required for the AP location and can possess a different role when binding to Atg18 in order to target the complex to autophagic membranes [79]. In Sz. pombe, depletion of nitrogen from the culture medium triggers autophagy. Unlike mammals, the addition of AMPK does not induce autophagy, nor can rapamycin induce it. It has been reported that rapamycin has a partially inhibitory effect on TOR activity, moreover, the addition of rapamycin plus caffeine prompts a starvation-specific gene expression pattern and autophagy [80–81]. Atg 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 18a, 18b, 18c, Vps34 homologues have been found (Table 2) [73–75], and their deletion inhibits AP formation.
4.4. Apoptosis
The maintenance of homeostasis in pluricellular and unicellular organisms is achieved through lots of mechanisms, one of the most important of which is related to the death of the cell itself. In this way, cell death regulates the number of cells in a tissue or a colony, and the removal of damaged cells. In 1963, Lockshin introduced the term Programmed Cell Death (PCD) in order to define the programmed and controlled self-destruction process in a local and temporal way [83–84]. There are many ways for a cell to die; one of them is via apoptosis. The term apoptosis was introduced by Kerr and colleagues in 1972, which is defined as a highly coordinated cellular suicide programme controlled principally by zymogens (i.e., caspases and metacaspases), proteins of the Bcl-2 family, and mitochondria [85]. The Nomenclature Committee on Cell Death established the morphological and biochemical changes in the apoptotic cell, such as membrane blebbing, cell shrinkage (pyknosis), chromatin condensation plus fragmentation (karyorrhexis), formation of membrane bound cell fragments (apoptotic bodies), decrease in mitochondrial inner transmembrane potential, selective cleavage of various cellular proteins, and the translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane [86].
The importance of Bcl-2 proteins relies on Cyt C, which in basal conditions works at the respiratory chain but plays a different role in the cytoplasmic space: Cyt C oligomerizes with the adaptor protein Apoptosis protease activating factor-1 (Apaf-1), forming the complex known as apoptosome [98, 99]. The apoptosome activates by proteolytic cleavage to caspase-9 which is one of the cascade cysteine-aspartate proteases (caspases); in other words, caspases are liberated from their inhibitory prodomain [98, 99]. Once caspase-9 is activated, the downstream zymogens procaspase 3/7 are activated as caspase 3/7, then they attach to specific substrates in the cell leading to cell dismantling but they must first be released from their endogenous inhibitor X-linked inhibitor of apoptosis (XIAP). XIAP activity is inhibited by SMAC/ DIABLO and HtrA2/Omi, which should be remembered as mitochondrial proteins that were released along Cyt C [94, 95, 100]. In C. elegans the mitochondrial permeabilization and the release of Cyt C have no role in CED-3 activation, unlike its mammalian counterparts. Another difference between the nematode and mammals is that CED-4 lacks a Cyt C interaction motif, this means that some of the apoptotic machinery are conserved among them. However, some protein-protein interactions differ from each other.
On the other hand, the extrinsic pathway start when death ligands such as tumour necrosis factor (TNF), Fas ligand (FasL), TNF-related apoptosis-inducing ligand (TRAIL), among others, join and activate their respective transmembrane death receptor, such as Fas. The interaction of death ligands with death receptors result in the recruitment of adaptor proteins like the Fas-associated death domain protein (FADD), which recruits, aggregates, and promotes procaspase-8 activation. Caspase-8 switch on procaspase 3/7 and the Bid protein through is proteolysis. At this point, a crosstalk between the extrinsic and intrinsic pathways are mediated by Bid activation since truncated Bid (tBid) promotes Cyt-C by interacting with Bax, leading to Bax insertion in the mitochondrial membrane as an oligomer pore [93–95].
Study of apoptosis is difficult in higher eukaryotes due to the complexity of the phenomena itself, which is why it was thought until recently that unicellular organisms could not have PCD machinery because it would mean that the organism could orchestrate its suicide, so yeast were employed as purely naïve backgrounds for studying proteins' interaction. In 1997, B. Ink and JM. Jurgensmeier independently performed a yeast two-hybrid system in Sz. pombe to study the proteins of the apoptotic cascade. It was found that the expression of the proteins Bax and Bak lead to the induction of chromatin condensation and DNA cleavage [101–102]. Moreover, the lethal phenotype was obliterated by the co-expression of the antiapoptic Bcl-XL and Bcl-2, therefore, yeasts undergo mammalian-like apoptosis. Likewise, C. James proved that CED-4 expression in the fission yeast induced chromatin condensation and it was abolished by CED-9 interaction [103]. Induction of a yeast apoptotic programme can be triggered by exogenous and intrinsic stresses, such as low doses of H2O2, acetic acid, valproic acid, HCl, and NaOH in the media, cell ageing, and temperature stress [104–107]; these stimuli induce ROS that also enable an apoptosis programme. Additionally, DNA damage-responsive proteins are highly conserved among fission yeast and metazoans; hence, defects in DNA replication promote high levels of ROS, promoting apoptosis. Interestingly, SpRad9 — the orthologue of the human Rad9 — controls cell cycle checkpoints, and also possess a BH3 domain which enables binding with Bcl-2 and Bcl-XL in mammal cells, prompting cell death. This proves that the Bcl-2 family of proteins may be evolutionarily conserved from fission yeast to mammals [108–112].
Caspases are the main components of the apoptotic pathway, and are not found in Sz. Pombe; however, it possess an orthologue in the metacaspase PcaI (Figure 4). Unlike mammalian caspase activation, PcaI undergoes auto proteolytic cleavage activation just as in metazoan caspases; however, until now there has been no evidence of any substrate of PcaI [104, 105, 113]. Notwithstanding, overexpressing PcaI in Sz. pombe does not induce an apparent cell death phenotype, moreover it appears to stimulate growth. Also, PcaI expression was up-regulated by oxidative and heavy metal toxicity, heat, hyperosmotic stress and DNA damage conditions, H2O2, sodium nitroprusside, and CdCl, giving cytoprotection; this evidence suggests that the metacaspase could be involved in growth and stress adaptation of Sz. pombe. It has also been demonstrated that the level of oxidized proteins is proportional to the presence of the metacaspase, possibly due to a cleaning function within the population. Apparently, PcaI has pro-survival and pro-apoptotic roles and further investigation is needed to understand its function [105, 113, 114]. The Htra2/Omi-like protein Nma11p (Nuclear mediator of apoptosis) is found in S. cerevisiae, which is a serine protease, having a pro-apoptotic function, and which aggregates in the nucleus after cellular stress conditions; in Sz. Pombe, its putative homologue has been found [115]. In mammals, an apoptotic marker is the release of CytC; as in yeasts, its presence in the cytoplasm is found after the induction of the apoptotic programme. Just as in mammals, the release of CytC decreases mitochondrial inner transmembrane potential, but unlike metazoans the upstream and downstream effectors of PcaI have not been elucidated. The release of CytC has been correlated with the induction of cell death in S. cerevisiae [116], which correlates highly with the mammalian intrinsic pathway; nevertheless, CytC release has yet to be investigated in Sz. pombe.
5. Sz. pombe: Heterologous expression systems of proteins
Fission yeast is a useful system for studying the function and regulation of genes, and an excellent host for heterologous expression of molecules derived particularly from higher eukaryotes because it produces high cell densities, short fermentation times, and the use of chemically defined media [117]. Difficulties in the production and purification of heterologous proteins are related mainly to proteolytic degradation of gene products by specific proteases of the host [118]. This makes it highly important to obtain strains that have specific mutations eliminating the activity of proteases. Success of this expression is based on the use of genome-reduced strains with the deletion of unnecessary genes. Gene-deletion technology used for this purpose is the LATOUR (latency to universal rescue) method, which eliminates a DNA segment of more than 100 kb [119, 120]. This strategy of gene deletion is useful for identifying genes that are essential to the yeast [121]. The Sz. pombe proteolytic system has been seldom studied. Currently, two proteolytic systems have been identified with different subcellular localizations: a vacuolar one (where can be found the Proteinase yspA, Proteinase yspB, Carboxipeptidasa Y1p, Aminopeptidase yspI, Dipeptidyl aminopeptidase DPK, and DPC) and another Cytosolic level (Leucin Aminopeptidase yspII, peptidase multicorn, and proteasome 26S) [122, 123]. From full-genome sequence data which are deposited in the MEROPS database, an output of 121 peptidases and probable peptidases, being non-peptidase homologues 39 proteases, has been identified [124].
6. Conclusions
This chapter discussed some topics related to the employment of Sz. pombe as a model system for the study of the cellular processes of eukaryotes. Despite the complexity of mammalian cells with respect to single-celled yeasts, many intracellular processes are highly conserved, such as cell cycle, autophagy, and ageing; there are even similarities at the level of the mechanisms of the processes carried out in the cell. In some cases, there is only the sharing of some genes, as in the apoptosis process. However, their genetic regulation could be similar in triggering the specific cellular responses. The use of yeast Sz. pombe as a model of study allows the generation of new knowledge with direct application to mammalian cells such as human ones.


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This crossword clue is for the definition: Muck partner.
it's A 12 letters crossword puzzle definition.
Next time, when searching for online help with your puzzle, try using the search term "Muck partner crossword" or "Muck partner crossword clue". The possible answerss for Muck partner are listed below.
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Possible Answers: MIRE.
Last seen on: LA Times Crossword 6 May 2018, Sunday

subset: commoncrawl


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If someone likes (like I do) old way of ending call with hardware button you can use this old qwerty.kl I extracted it from one of older TBD ROM's (it is attached - QwertyOLD.zip)... You will loose Advanced Power Menu but you can install Quick Bo...
Can you share please how did you flashed vboot,wboot partitions? Here you are, attached files I modified for flashing appropriate partition ;) I cannot use xboot and yboot so these files are currently unusable but for future use maybe usable ;)...
I managed to have triple boot system ;) Booting Tytung's 2.7 build from NAND and booting AOKP and PA from SD-EXT :D:cowboy::D:laugh::victory::victory::victory: And all of them are faaaaaast :highfive::good::fingers-crossed::victory: First of all ...
I could earn little money when you finish downloading files from the file hosting sites I provided. I know the download speed of these sites are slow and may waste your time. However in my viewpoint, I also wasted my time for developing this new ...
Hi folks, Since I got a couple of private messages about my absence lately.. I just wanted to let you all know that I am fine, still alive and still have my HD2 though it's acting up more and more lately.. It seems the famous 'non responding tou...


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Technical Paper
Effect of Strain Rate on Mechanical Responses of Jute-Polyester Composites
There has been a keen interest in recent times on implementation of lightweight materials in vehicles to bring down the unladen weight of a vehicle for enhancing fuel efficiency. Fiber-reinforced composites comprise a class of such materials. As sustainability is also a preoccupation of current product development engineers including vehicle designers, bio-composites based on natural fibers are receiving a special attention. Keeping these motivations of lower effective density, environment friendliness and occupational safety in mind, woven jute fabric based composites have been recently studied as potential alternatives to glass fiber composites for structural applications in automobiles. In the past, mechanical characterization of jute-polyester composites were restricted to obtaining their stress-strain behaviors under quasi-static conditions.


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Duration: 2hrs
Price: £28
Contact: Angela at Sunlight & Blue Cyanotype & Lampshade Workshops
Course Start Dates
Course dates are flexible; please get in touch for the next available date.
About the Course
The Cyanotype photographic printing process was used in the 19th century by architects, engineers and scientists to copy drawings. By mixing Ferric Ammonium Citrate and Potassium Ferricyanide with sunlight, beautiful blue images are created. It is such a magical process and people are always really pleased and excited with their results. NB. the chemical is odourless and harmless when used under normal conditions.
Using the Cyanotype process, you will learn how to light-sensitise watercolour paper and fabric, place objects on top and then expose to light to create blue prints. It is an amazing and very satisfying process! You will get interesting results every time, and you may then want to progress onto my Cyanotype lampshade or Cyanotype silk scarf workshops.
I have been teaching photography to students of all ages in both formal and workshop settings for over 20 years. A particular love of mine is the exploration of old traditional processes, such as silver gelatin printing and cyanotype printing in which you create your own light sensitive surfaces.
The cyanotype is such a fun and rewarding process and you don't need any special skills or equipment...just a love of the colour blue!
To arrange a Cyanotype printing workshop, contact me to arrange a mutually acceptable time and location. This is a process that needs good UV light levels, so will take place from Spring through to Autumn.
I hope to see you soon.
No previous skills required for my rewarding short workshops that include all materials. I keep my groups small (3-6 people) which means that you will receive lots of help if you need it.
Angela Heron Watkins
''Angela is so generous with her skills and materials. I learnt so much and enjoyed every minute. I would highly recommend her workshops to anyone, complete beginner to professional artist." Pam Du Val May 2018
If booking with a CraftCourses voucher for part or all of the course fee, please fill in the contact form above. Your voucher code must be provided at the point of booking.
What's included in the price?
All materials & equipment are included
I will provide refreshments.
You need to bring a selection of plant material, and an apron to protect your clothes.
Even the drive to Angie's cottage near Weymouth was idyllic and only the beginning of a wonderfully inspiring day. For some time I had been thinking of taking a course in lampshade making and in cyanotyping; to find them both wrapped up in one course for a very reasonable price was very exciting.
Angie asked us to gather plants and natural material to bring with us and, after talking us briefly through the history of cyanotyping, including its use as the method for making blueprints, we got to work with several trial pictures, placing our natural material on to chemically treated light-sensitised watercolour paper and taking them to develop in the sun in Angie's garden. The results were really inspiring and I was surprised that I had been able to create something that looked that beautiful.
We progressed to making prints on light-sensitised material, in readiness for making the lampshade. After a trial run, it was time to create the final piece of indigo-coloured material for the lampshade. Making the lampshade seemed easy because Angie gave each of us her individual attention. It was clear that no-one was going to fail on her course and with a gentle approach guided us each to success.
I went with my mother and we both enjoyed the day so much. Angie is quietly spoken and a good and inspiring teacher.
The nice thing about cyanotypes is that the method produces results you are not able to foresee. The indigo background together with the pale blue or white shapes created by the plant materials make each creation inspiring and trendy. (One lady on the course brought some cut-out hare shapes which she used to great effect.)
This is an incredibly inspiring experience, exceptional value for money and at the end you get to take home your trial pieces on watercolour paper and material and your lampshade.
If money is an issue, you can make a small but beautiful tall tea light holder.
I would recommend this as one of the best Christmas or birthday presents for a friend, family member or yourself.
And, the wonderful thing about Angie's courses is that they generally take place on sunny days with a beautiful blue sky. Uplifting!
Claire Waring - - #26400
Response from Sunlight & Blue Cyanotype & Lampshade Workshops:Thank you so much for the wonderful review, Claire.
Spent a wonderful afternoon making a lampshade and many experiments with lengths of paper and fabric. We learnt about the technique and with Angela's calm manner and encouragment, felt confident to have a go and try different objects and materials. We had so much fun and excitement when seeing what everyones efforts looked like. Some really surprising results. Highly recommended especially in a group.
Nancy Grace - - #24926
Relaxed informative and productive. Two hours of experimental workshop. I would certainly recommend.
Ian Coleman - - #24412
I spent a most enjoyable morning with Angela who organised everything we needed for the class, including the sun. I experimented on both paper and fabric and trialled a number of different objects - seed heads, photos, stencils - and different exposure times.
It's a fascinating technique which Angela explained extremely well. I look forward to further experiments.
Fran G - - #24411
I had been longing to do the cyanotype printing course for a long time and finally managed to do one this morning!
Angela was great, really took time to explain everything and come up with suggestions.
A great laid back atmosphere, and I'm happy to say I'm pleased with my results.
I will definitely be attempting this on my own!
Wendy Cockcroft - - #24125
This course was fabulous. It is hard to say anything new that hasn't already been said in the reviews above. Angela is an excellent teacher, she provided all the materials and had prepared well in advance. She explained the process very clearly and organised everything very well, (even the sunshine). I met two lovely people and we all went home with results we were pleased with. I have ordered the materials and I will be continuing to practice this process.
Jenny Guest - - #23573
Great fun and great results.
Angela explained the process well and this interesting workshop just flew by. Angela had prepared some photos for us in advance so we managed to do organic and photographic images. We came away with loads!
If you like active crafting and super results you'll enjoy this workshop.
Liz Gomme - - #17532
We were very lucky to have a bright day in September to do our Cyanotype workshop. Angela provided all the materials for our lampshades, we just had to provide the decoration. Everything was well organised and Angela was an excellent teacher - very patient and good fun. Lovely way to pass a morning with friends and you get a lovely, unique lampshade at the end of it.
Thoroughly recommend.
June Mitra - - #17506
Course dates are flexible; please get in touch for the next available date.
This craft course provider has been awarded Silver status for receiving 5 or more 5 star ratings from previous students.
The Students' Choice award ensures that course providers get recognised for their efforts in the teaching and practice of UK craft.
They also allow tutors to gain valuable feedback on their courses and for new students to find the course that's right for them.


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Awapuhi Wild Ginger Texturizing Sea Spray
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- Brand: Awapuhi Wild Ginger
SP Sophia Collection Duchess Daisy Floral Bouquet Crystal Rhinestone Stretchable Corsage Bracelet in Ginger Gold
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Spark a Royal Garden Party fashion buzz with these luxurious SP Sophia Collection Signature crystal rhinestone daisy floral bouquet stretchable fashion corsage bracelets ★ This unique posh design is perfected with pearlized daisy petals, a crystal solitaire center bulb, layered over a hobnob of larger crystal petals & encrusted rhinestones ★ Stretchable band, one size fits most ★ Available in 8 colors Gardenia Green, Begonia Blue, Poppy Purple, Forget-me-not Fuchsia, Ginger Gold, Sweetpea Scarlet, Peacelily Platinum and Apsen Amber ★ Pair with our matching detailed stretchable ring
- Color: Ginger Gold
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Mix Curcuma Bulbs -5 Bulbs- Ginger Curcuma Officinal Summer Flowering Prepared Bouquet Outdoor Decorative Landscape Balcony Tubers
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Curcuma bulb is named for its beautiful inflorescence and its pink bracts resembling lotus. At the same time, it is called "tropical tulip" because of its tropical origin and tulip-like flower appearance. Perennial bulbous herbal flowers. Plant height is 30-80 cm, flower diameter is usually higher than leaf. There are spindle-shaped or spherical rhizomes in the ground. There are two rows of opposite buds on the corm. The growth of buds has the habit of biasing to one side, which can produce many successive buds. But after planting in spring, the first generation of buds usually germinates first. This bud usually has 5-7 leaves, and then only 3-4 leaves grow.
- Color: Mix
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Glamology Proven Ayurvedic Mahanarayan Therapeutic Moisturizing Body Massage Oil - Herbs with Coconut Oil, Sesame Oil, Turmeric & Ginger for Sore Muscles & Stiffness
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Melt away all the stresses and frustrations of the day with Glamology Massage Oil, an aromatic bouquet of soothing essential oils and nourishing botanicals work in harmony to pamper your skin and senses. Let the calming combination of Coconut Oil, Sesame Oil, Turmeric Ginger help you unwind, relax, and recharge and hydrate your skin for a silky soft feel. You can also use this oil to perform a relaxing or sensual massage to ignite romance and passion.Expertly Blended Massage oil allows your hands to glide easily resulting in an incredible massage. Best Massage Oil for Therapeutic Massage, Relaxing Massage, Body Massage, Sports Massage, Deep Tissue Massage, Hot Stone Massage and more. All Natural, Great for Sensitive Skin, Hypoallergenic Paraben Free. Made with the Best Oils for massage. This oil is the perfect consistency, not to heavy or to light. Glamology Therapeutic Massage Oil won absorb to quickly and will leave skin perfectly smooth rather than greasy and sticky. Thanks for Helping Children.As a part of our philanthropic efforts 15% of the sales proceeds are donated to Schools where children are given free education. Over 2 million Dollars have already been donated.Make a Difference in a Child's life. !!!
- Brand: Glamology
INDIAN SPLENDOR Kashmir Masala - Exclusively Handpicked, Premium Assam Black Leaf Tea with Premium Spices- Cinnamon, Cardamom, Ginger, Cloves, Liquorice, Fennels and Pepper. (Robust and Rejuvenating)
INDIAN SPLENDOR Kashmir Masala - Exclusively Handpicked,
Kashmir Masala is a very special, Robust and Rejuvenating, Indian splendor blend. It has a combination of brisk Assam Granular tea leaf with a bouquet of pure, natural and healthy Spices. Truly Refreshing aroma and flavor. A connoisseur's delight. Double packed, first in sealed foil pouches to keep the tea and spices garden fresh, and then in Designer Metal Caddies for additional preservation and Classic presentation.
- Brand: INDIAN SPLENDOR
Home Comforts Peel-n-Stick Poster of Tropical Vase Flowers Ginger Still Life Bouquet Vivid Imagery Poster 24 x 16 Adhesive Sticker Poster Print
Home Comforts Peel-n-Stick Poster of Tropical Vase Flowers
We stand behind our products and services to deliver to your doorsteps within the promised delivery window. We appreciate your business for any questions or concerns please contact via email.
- Color: Multicolor
- Brand: Home Comforts
Gingerbread House Lollipop Bouquet by Escape Concepts
Endangered Flora of Hawaii and visit a family farm to gather a colorful bouquet of tropical blooms
XDCGG Car Window Shade Small Cute Ginger Cat Bouquet Red Tulips 55x30 Inch Anti-uv Coating Protect Seats Foldable Polyester and Aluminized Film Sun Shade Car
XDCGG Car Window Shade Small Cute Ginger Cat
☀Material:Polyester and Aluminized Film☀Size:55inch x 30inch (140cm x 75cm)☀With the car sunshade there is no need to worry about burning steering wheels, scorching dashboards, or hot car interiors. You, your children, and pets can enter your vehicle with peace of mind that it won't feel like an oven.☀UV rays can crack, fade, and damage your car's interior. The car sunshade acts as a 1-piece protective UV sun shield that helps ensure your interior looks good for years to come.☀Our most popular size is great for large size windshields found in SUVs, trucks, and cars.
- Brand: XDCGG
Bouquet in a Persian Vase Acrylic Painting with Ginger Cook
Once again Ginger cook introduces another ODG (old dead artist_ ) whos painting delight an inspir...
Bouquet of Wild Flowers Beginning Acrylic Painting Tutorial
How to paint wild flowers with a blue vase is a full length 16x12 step by step video that featur...
How to Paint a Bouquet of Flowers Floral arrangement by Maximilian Luce
Maximilien Luce was an impressionist painter at the turn of the century. His Bouquet of Flowers i...
Ginger & Spice a Florist in London offering Flower Bouquet
Fulfilling all your floral design dreams! Welcome to Ginger & Spice...Creating you the best desig...
Tropical Arrangement By Everyday Flowers
http://www.everydayflowers.net/Tropical.html. One of our Tropical Arrangements Featured on Our W...


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Congratulations to Xi Cao who won the best poster presentation at the ChemCYS 2016 conference last week in Blankenberge, Belgium. The poster was on her research on "Separation of Organophosphate Nerve Agents by Capillary Electrophoresis and Microchip Capillary Electrophoresis". This research is funded through the GIFT project under the FP7 Security Programme.
ChemCYS 2016 is a Chemistry conference for young scientists and this year had Nobel Laureate Prof. Dr Sir Harold Kroto, The Florida State University as the keynote.
GIFT is a Capability Project funded under the FP7-SEC-2013-1 call to develop a European toolbox, focusing on procedures, practice and guidelines for CBRN forensic aspects. The title of project is Generic Integrated Forensic Toolbox for CBRN incidents. Full details of the project can be seen here https://giftforensics.eu
The objective of Xi Cao's work can be seen below
"Forensic investigation is a key component in the fight against crime and the protection of EU societies. At present it is hampered by a lack of protocols and training in carrying out forensic analysis on CBRN-contaminated materials. The aim of GIFT-CBRN is to develop a forensic toolbox for investigating CBRN incidents. This toolbox would provide (1) procedures, sampling methods and detection of CBRN agents at the crime scene, (2) traditional forensic laboratory methods for contaminated evidence and (3) laboratory methods for profiling the CBRN agents released at the incident. The procedures and methods are will be set up and validated according to ISO17025 and the system validation will be performed by a final exercise. Procedures for chain of custody, QC to ensure the integrity of the evidence and investigations done on the evidence from crime scene to court will be developed. An education and training curriculum related to the developed procedures, best practices and methods will be designed and progressed to implementation.
Underpinning the above aims, research will be carried out to develop novel methodologies to enable traditional forensic science (DNA, fingerprint and electronic devices) to be carried out on CBRN contaminated exhibits and analytical procedures to be carried out that not only provide information about the CBRN agent itself but also through CBRN profiling provide in-depth information which can give valuable forensic information, for example points of origin.
The project team includes forensic research laboratories, potential users of the expected developments, including public and private users, and SMEs who will be able to bring the new technologies developed within the project to market."


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Events in and around Jindabyne..
Snowy Ride - 2/11/19
The Snowy Ride is a motorcycle event run annually in the Alpine Region of NSW since 2001 and has helped the Steven Walter Children's Cancer Foundation raise in excess of $8 million for research into a cure for childhood cancer.
Snowy Mountains Inaugral Trout Festival - November 2019
The committee hopes you enjoy fishing the beautiful Snowy Mountains area.
Mountain Bike Opening Weekend - 16/11/19
Dust off your bike park weapon of choice, grab your mates and hit the Thredbo Mountain Bike Park for first laps of the season. The trail crew will have spruced up the expanding trail network and added some single track with Woody's making its debut.
Alpine Hiking Season Open - 16/11/19
Experience the natural beauty of Australia's highest mountains in summer as you walk and hike some incredible alpine trails. Whether it's a family friendly hike to the top of Mt. Kosciuszko or an advanced day hike across the Australian Main Range, there's something for every age and ability to enjoy.
Le' Tape Australia - 29/11/19 to 30/11/19
Since its inception in 2016, L'Étape Australia by le Tour de France has provided a new level of riding experience in Australia, combining the beauty of the Snowy Mountains, the incredible enthusiasm of the locals and an event run to Tour de France professional standards.
Wild Flower Season Starts - 30/11/19
The Australian Alps come alive in summer with mountains of meadows for you to explore. December – February is the best time to grab a picnic, head high and watch the wild flowers explode with colour.
Cannonball Mountain Bike Festival - 4/12/19 to 8/12/19
Australia's biggest gravity inspired mountain bike festival returns in 2019 setting the standard for mountain bike competition with a huge line-up of supreme events and non-stop entertainment all weekend long.
School Holiday Events - 21/12/19 to 11/1/20
The School Holidays Adventure festival in Thredbo has a mountain of activities the whole family can enjoy. Treasure hunts, bollywood dance sessions, kids mountain bike races, arts and crafts, rock-climbing, face painting and lots more….
Man From Snowy River Rodeo - 28/12/19
The annual Man From Snowy River Rodeo event has been running since 1943, originally in Old Jindabyne and now at John Connors Oval, Park Road, Jindabyne Sports Ground.
Ney Year's Eve Concert - 31/12/19
The Thredbo New Year's Party is a great way to welcome 2020, with a big party and entertainment for all ages.
Australian speed and marathon water ski championships - 8/1/20 to 12/1/20
Held on Lake Jindabyne.
Thredbo Blues Festival - 17/1/20 to 19/1/20
The Thredbo Blues Festival is bursting with talent in 2020 for its 26th year with another amazing program of exceptional Australian and International blues players getting up close and personal in a music festival like no other.
Australia Day BBQ on Mt Kosciuszko - 26/1/20
Thredbo is inviting Aussies to celebrate Australia Day at the highest point in Australia, on top of Mt Kosciuszko 2228m.Perfect for the family or a long weekend away with mates, Thredbo will be taking Australia Day festivities to new heights celebrating with an exciting line up of iconic Aussie entertainment and activities, offering an unforgettable Australia Day!
Kosciuszko Craft Beer Festival - 7/2/20 to 8/2/20
Celebrate the art of craft beer brewing this February at the Kosciuszko Craft Beer Festival in Thredbo.
Australian Alpine Accent - 8/2/20
he Australian Alpine Ascent (AAA) Festival is specifically designed to be the world's most gruelling set of single day challenges held within daylight hours. The festival includes two trail runs 25km and 50km.
Snowies Mountain Bike Festival - 14/2/20 to 16/2/20
The Snowies MTB Festival is back in 2020 with the iconic Snowy Mountains playing host to the ultimate mountain bike experience.
Valentines Day - 14/2/20
Treat your loved one to a long weekend away.
Trail Run Australia - 1/3/20
The Snowies Trail Run Festival is hosted in conjunction with the TreX Cross Triathlon Series which together make up an action packed weekend of off road events for the entire family.
Thredbo Food & Wine Festival - 6/3/20 to 8/3/20
Chase a 7kg cheese wheel to glory, thrills and spills for all! The champion male and female will take home a 2020 Thredbo winter season pass! This year the Cheese Rolling Competition also sees the best fancy dress competitor winning a voucher.
Lake Light Sculpture held by Lake Jindabnye - 10/4/20 to 13/4/120
The Easter long weekend offers a fantastic opportunity to experience Jindabyne. The sculptures are displayed in an evocative setting with the reflections of the Snowy Mountains on the lake, providing a living studio for the artworks but it is as night falls that the real magic begins. Illumination has, since the festival's inception, been a defining element of Lake Light Sculpture, and the sculptures are lit from sunset to 10pm each evening.
Mothersday - 10/5/20
Show your mother you care with a weekend getaway!
School Holiday Events - 11/5/20 to 25/5/20
The School Holidays Adventure festival in Thredbo has a mountain of activities the whole family can enjoy. Treasure hunts, bollywood dance sessions, kids mountain bike races, arts and crafts, rock-climbing, face painting and lots more….
Peak Music Festival at Perisher - June Long Weekend 2020
Peak Music Festival kicks-off the start of the Perisher ski season on June Long Weekend with 4 days of cool music, snow, fun and laughter.


text: In this quick video clip Colin will show you how to draw a realistic African Serval Ear. You'll learn how to layer colours from light to dark using Pastel Pencils to create stunning effects. The pencils he uses are Faber-Castell Pitt Pastel Pencils and on this occasion Colin is trying the Canson Mi-Teintes Touch Pastel Paper. Watch the 8 minute lesson below:


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This SEBO 9' Stretch Exhension Hose connects to the attached hose on their uprights plus to extend the total hose length. This hose is great for reaching high areas and cleaning stairs while safely keeping the upright at the bottom. View Larger Image
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text: I know it creeps up on you every year and then BAM, it's tax time again. We are available for just one time questions if you need it or a boost of time to sit down and wade through your piles of papers. Let us know where the issues are and we will try our best to get everything ready to roll for tax time.


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1099: Where is my 1099 from 2018
I'd be glad to share with you some information about your 1099 form for 2018.
If your client submits your final form to the IRS, we'll notify you to view your 1099 online via email. Otherwise, I recommend reaching out to your client for the details of your 1099. This way, […]
Resolution for '1099 issue: 'Where is my 1099 from 2018' available: Yes.
The 1099 issue or error code is a known issue in Quickbooks Online (QBO) and/or Quickbooks. Support for this issue is available either by self-service or paid support options. Experts are available to resolve your Quickbooks issue to ensure minimal downtime and continue running your business. First try to resolve the 1099 issue yourself by looking for a resolution described in the article. If it is a complex 1099 issue or you are unable to solve the issue, you may contact us by clicking here or by using other 1099 support options.
For more information about resolving issues related to 1099 in Quickbooks/QBO or to get support for the issue 'Where is my 1099 from 2018', click the link below:


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Football Brawl Prompts Off-Duty Cop to Fire Shots
Things got chaotic on Monday night at the Isidoro Beaton Stadium in Belmopan. A football match was underway between Galen University and the University of Belize when a fight broke out between players from both teams. Seconds after, an-off duty police officer is caught on camera pulling out a firearm and blasting two shots in the air. His actions were intended to stop the brawl, but it was futile. It has instead gotten him and two other police officers in trouble and the Professional Standards Branch is said to be looking into the matter. The fight is being investigated by both universities. Hipolito Novelo reports.
Hipolito Novelo, Reporting
Shots were fired by an off-duty police officer during an all-out brawl which broke out at an ATLIB football match at the Isidoro Beaton Stadium in Belmopan on Monday night. Cell phone footage of thirty-five seconds shows football players from the University of Belize and Galen University attacking each other. In the black uniforms are the UB Black Jaguars while the Football Galen Eagles wore the orange-red uniform.
Insults were hurled. Punches were thrown and even kicks were fired. It was a chaotic mess that even off-duty officers were not able to control.
This off-duty officer runs towards the sparring crowd. He pulls out a firearm. Meanwhile, from the bleachers, this other person also believed to be an off-duty officer is seen pulling out a firearm from the waist of another man believed to be an off duty officer. He too runs towards the crowd with the gun in the air. Meanwhile, the first officer points the firearm in the air and fires two shots. This, however; fails to quell the angry players.
Both universities have launched an internal investigation.
Prof. Dr. Clement Sankat, President, University of Belize
"The University of Belize together with Galen our sister university, together with the ATLIB sports authorities, this matter is under investigation. It will be very difficult for me to speak to it because at this time we are not fully aware of all that transpired there. What we know is that the gunfire was not from our students. They were not involved in that and neither the Galen students were involved in any use of guns Et cetera. We are clear about that."
A joint release from both universities states that they will collaborate with each other and the ATLIB sports authorities in its investigation and disciplinary process.
Prof. Dr. Clement Sankat
"The investigation wills also possibly lead to any disciplinary procedures that we have in place if that becomes necessary. I want to assure and the public that neither of our universities condones any kind of violence in any of our activities. And if students have breached their codes of conduct, both universities will do what is necessary. I want to assure you having investigated this matter we will want to ensure that a repeat does not occur."
"Is there the possibility that students can file a criminal complaint against each other seeing that there is assault in the video?"
Prof. Dr. Clement Sankat
"All citizens are entitled to that. You are not only a student of the University of Belize. You are a citizen of Belize. You can file your own complaint with the police. My hope is that it does not escalate to that. That our universities will find a way to investigate this and to dispense the kind of penalty that suits the kind of misdemeanor or the thing that happened out there. Let us not prejudge the issue."
The Football Federation of Belize says that it will monitor closely the outcome of both current University investigations. Based on the outcome of the investigations any player found at fault registered with the F.F.B. will be dealt with accordingly. Concerns have been raised by the actions of the off-duty cops. Those who have seen the video believe that the officers acted recklessly. We understand that the matter has been referred to the Professional Standards Branch. Hipolito Novelo, News Five.
The UB Black Jaguars defeated the Football Galen Eagles four to two.


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Quiet Owl Late Night e-liquid
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Suitable for drippers, Suitable for sub-ohm vaping
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2019.09.07 Hua Chenyu Running Shark Music Festival, Chengdu China>Composer: Hua Chenyu Lyrics: Ding YanXue Arranger: Bernard Zheng, Hua Chenyu Producer: Bernard Zhang Translated by Hua Chenyu English Subs Please subscribe for more translated videos of Hua Hua River Child=Water Tiger in Daoism La Vi Ah Vi Ah Vi Night arrives Continue Reading
Drake, Grand View pick up wins
Saturday for Drake: no offense, but no problem the bulldogs defense was lights out against Truman State. Rick Fox looking for his t frost win as head coach. Tied at 7 – secodn half. Held truman state to 290 total yards. Caz Zyks lowers the boom on t his play! Continue Reading
Dickens Style Christmas Carolers – Corporate Entertainment – 2019
Dickens style Christmas Carolers for hire Book your Dickens Style Christmas carolers Our Holiday Carolers can come dressed as Dickens Style Christmas or Holiday carolers Book today Don't Wait Contact us at
Jesus Christ Vampire Hunter (FULL MOVIE)
Ele te ve, Ele te ouve… Ele ve e sente todos seus pecados. Todos seus horriveis e humilhantes pecados. Você está aí Deus? Por que não me respondes? Por que não me dá um sinal? Me responda! Por que estou aqui? Eu não O vejo! Mostra-me que Se preocupas comigo! Continue Reading
Basic Animals ASL
Hello! This is Wakewolf *sign name* I have a list of animals sign Are you ready and are you excited? That's all for now I will make more list later. Enjoy the video and I will make ASL gif. Enjoy to watch and learn! Have fun! Don't forget to hit Continue Reading
Hustler Elite | Get Trained by Tony Hustle to Become a Master Seducer
okay guys Tony hustle here happy 2019 you know my rule is if you want to get out the banana you have to write the banana so we're gonna throw in a little banana too right now enjoy the right you saw I'm going to make an announcement today we're Continue Reading
Empiezo un mundo nuevo en minecraft Busco recursos pa' hacerme una casa Rompo madera, para hacer la mesa Dónde se craftea y poder hacerme una espada Un juego arriesgado en supervivencia Otros jugeos se han copiado, es una evidencia Hasta me entretiene matar una obeja Por mucho que pase, no Continue Reading
o matador de carangueijo
o matador de carangueijo
Matador News Episode 2: College Tuition on the Rise
Good morning all Matadors. This is Jizzele Zelaya reporting live from Cal State Northridge and this is Matador News. The rise of college tuition occurs here and then but does it affect the student's productivity? In fact more college drop outs occurred due to financial instability compared to cases that Continue Reading
Lots of Cows Goats and Sheep Toy Collection
Welcome to racetoytime! Hi kids, racetoytime here today I'm going to show my complete Schleich cattle sheep and goat collection. First I'm going to show you the holstein cows here they are I'm going to show them to you one by one these are my Jersey cows we have three Continue Reading


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Galaxy introduction for life scientists - streamed or live
|17 June 2020
|Lucille Delisle (EPFL) and Hans-Rudolf Hotz (FMI, SIB)
|Cancellation deadline
|3 Jun 2020
Should this course be streamed, it will be only for registered participants.
Galaxy is an open, web-based platform for accessible, reproducible, and transparent computational biomedical research. Galaxy gives you access to thousands of tools. For example, you can run a complete NGS analysis from fastq files to publication-ready figures using only your web browser.
This course will give a general introduction on the galaxy web-page structure, how to import data, run tools, share analyses. Participants will run a whole NGS analysis using an ATAC-seq dataset as an example.
This course is targeted to any life scientist who wants to learn more about the web-based Galaxy platform and would like to discover its potential to analyze NGS data.
Learning objectives
After the course, participants should be able to use a public Galaxy server for their research and be able to:
- reproduce and share an analysis in Galaxy
- run tools such as FastQC, bowtie2 and macs2
- understand their outputs
- interpret the biological results obtained with Galaxy
Knowledge / competencies
No command line experience or programming knowledge is required. However, familiarity with NGS and genomic data is highly recommended.
A Wi-Fi enabled laptop with Firefox, Chrome, or Safari web browser installed. There will be access to the eduroam and guest-epfl networks.
The registration fees for academics are 60 CHF and 300 CHF for participants from for-profit institutions. This includes course content material and coffee breaks.
Deadline for registration and free-of-charge cancellation is set is set to 03/06/2020. Cancellation after this date will not be reimbursed. Please note that participation to SIB courses is subject to our general conditions.
You will be informed by email of your registration confirmation.
Venue and Time
EFPL, room 032, AAB building, Lausanne
The course will start at 9:00 and end around 17:00. Precise information will be provided to the participants in due time.
Additional information
Trainers: Lucille Delisle (EPFL) and Hans-Rudolf Hotz (FMI, SIB)
Coordination: Patricia Palagi
We will recommend 0.25 ECTS credits for this course (given a passed exam at the end of the course).
You are welcome to register to the SIB courses mailing list to be informed of all future courses and workshops, as well as all important deadlines using the form here.
For more information, please contact email@example.com.


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Today, April 29 at 4 PM in Prague, visitors will have the chance to join Prague residents in joining the nearly one-quarter of the world's people without shoes. One Day Without Shoes is set to draw attention to how poverty affects our world.
Bringing Eastern Europe closer to your heart. Sometimes we look at the world too.


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The fashion industry in Kenya is slowly growing. The middleclass is getting bigger and the
interest for fashion among these consumers is increasing. One major problem for the growth
of the industry is the extensive second hand trade, which has given several problematic
affects. The textile production has almost disappeared, the western influences are evident and
the consumer's price sensitivity is prominent.
The aim of this thesis is, by conducting a field study; to qualitative examine how to develop a
strong domestic fashion brand on the Kenyan market and to give a foundation for a branding
strategy for people involved within fashion in Kenya. Some of the major findings for this
thesis are; the excessive second hand trade and its negative affect on the domestic fashion
industry, the less developed fashion environment, the growing interest for fashion among
Kenyan consumers and the low credibility the consumers have for domestic brands.


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SpaceX has changed the design of the SpaceX Super Heavy Starship to stainless steel. However, the design changes are more complicated than just using stainless steel instead of carbon fiber or aluminum. SpaceX will not need advanced Pica-x ceramic heat shields in most of the rocket.
Elon Musk indicates that active cooling with super-cold fuel only has to reduce the temperature during re-entry by 300 degrees. The 310S stainless steel that SpaceX will use can repeatedly withstand ~1450 Kelvin.
SpaceX and Elon Musk have to make the materials, design and technology work. If they succeed using stainless steel they will massively cut the development time. Stainless steel is much easier and faster to work with than dozens of layers of carbon fiber. Success with this material and designs would provide SpaceX with another acceleration in development speed compared to their competition.
Elon Musk and SpaceX still have many skeptics. SpaceX now has the best track record for developing and launching new rockets with the multiple Falcon 9 version and the Falcon Heavy. I will assume that SpaceX has the data, the simulations, and physical testing to justify the design choices that are being made.
SpaceX and Elon Musk are far from perfect. They just allowed the Starhopper to be damaged in 50 mph winds which will cost weeks in repairs. However, United Launch Alliance (ULA) and Blue Origin have not launched new rockets to orbit in the last fourteen years. ULA has launched many older rockets. Blue Origin has had sub-orbital launches.
When going to ~1750 Kelvin, specific heat is more important than latent heat of vaporization, which is why cryogenic fuel is a slightly better choice than water
— Elon Musk (@elonmusk) January 22, 2019
310S stainless is better for high temp outer skin, as it can take ~1450 Kelvin, so active cooling with cryogenic fuel only has to mitigate 300 degrees of delta temp
— Elon Musk (@elonmusk) January 23, 2019
80% of the SpaceX Starship will not reach a temperature that will need a heat shield. Only 20% of the ship will need the active cooling system that SpaceX is building.
~1750K is peak heating expected on about 20% of Starship for LEO entry, ~1600K on 20%. Rest drops below 1450K, so no heat shield needed. Radiative cooling at T^4 takes care of 60% of the ship. Another reason for steel.
— Elon Musk (@elonmusk) January 23, 2019


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A Daily Word with Dr. Ed Young. Daily thoughts from God's Word that will help you find strength, resolve, peace and comfort. Truth For Life Daily, with Alistair Begg. Discover the best Teen & Young Adult Christian Devotionals & Prayer in Best Sellers. Find the top 100 most popular items in Amazon Books Best Sellers.
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Worldwide Devotional For Young Adults. Sunday, May 5, 2019. Elder and Sister Cook. Watch It | Biography | Poster. 2019 Schedule Jan. 13—Elder Dale G. Renlund; May 5—Elder Carl B. Cook.


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Roadhouse News - February 2002
February 2, 2000
I have decided to write this news edition myself to express my sincere thanks to someone who continues to inspire me, and to say the least, continues to open doors for me. Lee Roy Parnell my friend. His invitation to me to sit in with he and his band three years ago was definitely the match that lit the fire which had burned out long ago. You see, I burned out about the time Lee Roy was just coming on the scene. Actually I dont know if I should thank him or shoot him!
Since that night in San Antonio three years ago I have played countless Texas dates with Lee Roy. I have felt the live show adrenaline of fellow Texan, PAT GREEN, experienced the JAY BOY ADAMS BAND REUNION, been special guest along with Ronnie Dunn, Radney Foster, Bruce Robison and Kelly Willis at LEE ROYS Exit Inn Nashville/Texas Party, and toured on the 1999 GEORGE STRAIT COUNTRY MUSIC FESTIVAL TOUR, and IMUS IN THE MORNING backing up Lee Roy doing "On the Road". And now, I am going to Europe to tour with THE TEXAS TORNADOS featuring LEE ROY PARNELL. Am I fired up? Fired up would be an understatement! While checking out web pages take a look at www.dougsahm.com and bring yourself up to date.
Late winter of 1999 I played with Lee Roy in Wimberley, Texas. His show was an unplugged version. There were several special guests that night. Lee Roys brother, Rob Roy Parnell, myself, and of course Doug Sahm. After the show that night, I got to visit with Doug for about two hours. I had not seen him in years. He is everything and more that you have ever heard about Texas music. He is Texas music! And come to find out this show in Wimberly, Texas, is probably the last time Doug Sahm performed live. I was a part of that and again because of Lee Roy. Three weeks later Doug passed away. That night will remain a permanent part of my soul.
I actually underestimated, or maybe I just forgot if thats possible, how much music has played a part of my life. I will say this, it is beyond me how I just walked away and never looked back. That is... until I ran into Lee Roy Parnell... again.
Back Issues Of The Newsletter
Gallaway Issue | September
2000 - Dewitt Bender