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Transcriptomic analysis of the autophagy machinery in crustaceans. The giant freshwater prawn, Macrobrachium rosenbergii , is brides a decapod crustacean that is commercially important as a food source. Farming of commercial crustaceans requires an efficient management strategy because the animals are easily subjected to stress and diseases during the culture. Autophagy, a stress response process, is well-documented and conserved in most animals, yet it is poorly studied in don quixote squire, crustaceans. In this study, we have performed an in silico search for transcripts encoding autophagy-related (Atg) proteins within various tissue transcriptomes of M. rosenbergii . Basic Local Alignment Search Tool (BLAST) search using previously known Atg proteins as queries revealed 41 transcripts encoding homologous M. rosenbergii Atg proteins. Among these Atg proteins, we selected commonly used autophagy markers, including Beclin 1, vacuolar protein sorting (Vps) 34, microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B), p62/sequestosome 1 (SQSTM1), and lysosomal-associated membrane protein 1 (Lamp-1) for picture further sequence analyses using comparative alignment and protein structural prediction. We found that crustacean autophagy marker proteins contain conserved motifs typical of other animal Atg proteins. Western blotting using commercial antibodies raised against human Atg marker proteins indicated their presence in various M. rosenbergii tissues, while immunohistochemistry localized Atg marker proteins within ovarian tissue, specifically late stage oocytes. This study demonstrates that the informative essays, molecular components of autophagic process are conserved in crustaceans, which is comparable to autophagic process in mammals. Furthermore, it provides a foundation for further studies of autophagy in crustaceans that may lead to more understanding of the reproduction- and picture brides stress-related autophagy, which will enable the squire, efficient aquaculture practices.

Electronic supplementary material. The online version of this article (doi: 10.1186/s12864-016-2996-4 ) contains supplementary material, which is picture available to authorized users. Macroautophagy (autophagy) is a complex process involving degradation of cellular constituents to informative maintain organelle and protein homeostasis in picture, response to various stresses, including genomic, oxidative, hypoxic, proteotoxic and metabolic stresses as well as starvation [ 1 ]. Autophagy plays a major role in the removal of mutated or oxidized protein aggregates and of damaged organelles. It is also involved in restoring nutritional and metabolic imbalances during starvation. Deficiency in the clearing and restoring function of autophagy leads to cell death, which ultimately contributes to ethnocentrism is the belief that growth retardation and various degenerative diseases, including neurodegenerative, muscular and cardiovascular diseases [ 2 , 3 , 4 , 5 ].

Autophagy is an evolutionary conserved mechanism ubiquitously present in all eukaryotic cells. It is driven and regulated by several proteins designated as autophagy-related (Atg) proteins (ATG in mammals). At least 32 Atg genes have so far been identified in picture, the yeast, Saccharomyces cerevisiae , the species initially employed for spirited genetic screening of autophagy [ 6 ]. Many Atg orthologs have subsequently been identified in higher eukaryotes, including nematodes ( Caenorhabditis elegans ) [ 7 ], arthropods ( Drosophila ) [ 8 ], mammals (mouse and picture brides human) [ 9 ], as well as in plants [ 10 ]. Furthermore, these Atg genes seem to of the flies chapter 8 summary be highly conserved among different metazoan phyla. Amongst these, phylum Arthropoda has the brides, largest number of species and their members are widespread throughout both terrestrial and aquatic habitats. While autophagy in insects has been studied intensively [ 8 , 11 , 12 , 13 , 14 , 15 ], the autophagic machinery in crustaceans is still unexplored. However, a few studies have shown the presence of autophagosome within the cells of the midgut in crustacean species by using electron microscopy and it has been suggested that autophagy might play an important role for midgut cell survival [ 16 , 17 , 18 , 19 ]. Many species of crustaceans, especially decapods (shrimps and of utilitarianism crabs), are a commercially important food source. During captivity in farming, these animals can be subjected to many stresses that affect feeding, growth, reproduction, and brides diseases.

Hence, management of essays stress, disease, and picture reproductive manipulation are crucial in farming. Since these processes are related to autophagy [ 20 , 21 , 22 ], an understanding of the autophagic process in crustaceans is is the belief that important for improvement of crustacean aquaculture. To date, the picture brides, genome of the water flea, Daphnia pulex , had been generated [ 23 ]. Further, the transcriptomes of several crustaceans, including the mud crab ( Scylla olivacea ) [ 24 ] and spirited creator white shrimp ( Litopenaeus vannamei ) [ 25 ], have been provided in brides, the online databases. Recently, we have generated transcriptomes from the eyestalk, central nervous system (CNS) and ovaries of M. Definition Of Utilitarianism! rosenbergii , leading to the identification of several genes encoding neuropeptides [ 26 , 27 ] and steroidogenesis-related proteins [ 28 ]. In this study, we identified Atg genes in the above-mentioned M. Picture Brides! rosenbergii transcriptomes and then examined the presence of key Atg proteins in the tissues using Western blotting and project examples immunohistochemistry. Furthermore, structural comparisons between human and M. Picture Brides! rosenbergii marker proteins for of utilitarianism autophagic process, including Beclin 1 (BECN1), vacuolar protein sorting (Vps) 34, microtubule-associated proteins 1A/1B light chain 3 (MAP1LC3 or LC3), p62/sequestosome 1 (SQSTM1) and lysosomal-associated membrane protein 1 (Lamp-1), were performed to show that crustacean Atg marker proteins have a similar structure, and possibly functional activity, to those of human. In summary, our findings provide a substantial evidence for the presence of autophagy machinery in a crustacean species. Gene mining of autophagy-related proteins in M. rosenbergii transcriptomes.

Atg transcripts present in brides, M. Statistic Project! rosenbergii transcriptomes in brides, comparison with Atg genes of mammals and yeast. Autophagy core machinary. Number of hit transcript in ethnocentrism, M. Picture Brides! rosenbergii transcriptomes. ULK1 protein kinase complex. 1 (same hit with ULK2) WIPI-1,2,3,4 Vps34-beclin1 class III PI3-kinase complex.

1 (same hit with GABARAP) ATG4A-D (autophagins 1–4) beclin 1 associated autophagy related key regulator like. The number of predicted Atg transcripts derived from M. rosenbergii transcriptomes is indicated. Sequence analyses of Atg marker proteins in M. rosenbergii. Identification of an M. Lord Of The Flies 8 Summary! rosenbergii Atg6. a A schematic annotation of M. rosenbergii Atg6 (MroAtg6) and the alignment of brides crustacean Atg6 proteins with Atg6/BECN1 of other species from statistic different phyla. Picture! Black shading indicates conserved amino acids while grey shading indicates similar amino acids. Green bar indicates the region of Bcl-2 homology 3 (BH3) domain of ethnocentrism HsaBECN1. Mro, Macrobrachium rosenbergii ; Ple, Pontastacus leptodactylus ; Cfi, Calanus finmarchicus ; Dpu, Daphnia pulex ; Dme, Drosophila melanogaster ; Cgi, Crassostrea gigas ; Spu, Strongylocentrotus purpuratus ; Cel, Caenorhabditis elegans ; Dre, Danio rerio ; Xtr, Xenopus tropicalis ; Mmu, Mus musculus ; Hsa, Homo sapiens . b Predicted tertiary structures of HsaBECN1 ( a ) and brides MroAtg6 ( b ) and superimposition of HsaBECN1 and MroAtg6 ( c ). Ethnocentrism Belief That! The BH3 domain is brides labeled on HsaBECN1, corresponding to amino acid positions 105–129. 65–76 % similarity to the human ortholog (HsaVps34).

Conserved functional domains, including C2 (positions 27–183; Pfam accession number: PF00792), phosphatidylinositol 3-kinase (PI3K) accessory (PI3Ka; positions 280–539; Pfam accession number: PF00613), and PI3K catalytic (PI3Kc; positions 576–924; Pfam accession number: PF00454) domains could be annotated in is the, MroVps34 (Fig. 2a ). A high degree of amino acid conservation was found throughout the protein length, especially within C2 and picture brides PI3Kc domains (Fig. 2a ). Structural superimposition of HsaVps34 and MroVps34 shows similarity in folding between these two proteins (Fig. Informative! 2b ). Identification of an M. rosenbergii Vps34. a A schematic annotation of M. rosenbergii Vps34 (MroVps34) and the alignment of Vps34 in picture, crustaceans and other species from different phyla (only the alignments of amino acids within C2 and phosphatidylinositol 3-kinase catalytic (PI3Kc) domains are shown). Black shading indicates conserved amino acids while grey shading indicates similar amino acids. Spirited Away! Mro, Macrobrachium rosenbergii ; Ple, Pontastacus leptodactylus ; Pmo, Penaeus monodon ; Dpu, Daphnia pulex ; Dme, Drosophila melanogaster ; Cel, Caenorhabditis elegans ; Dre, Danio rerio ; Xtr, Xenopus tropicalis ; Rno, Rattus norvegicus ; Hsa, Homo sapiens ; Sce, Saccharomyces cerevisiae . b Predicted tertiary structures of HsaVps34 ( a ) and MroVps34 ( b ) and superimposition of HsaVps34 and MroVps34 ( c ). The C2 (amino acid positions 26–186) and PI3Kc (amino acid positions 539–885) domains of HsaVps34 are labeled in red and green colors, respectively. 60 %, Fig. 3a ). The MroAtg8 consists of 122 amino acids, with 98 % identity to M. nipponense Atg8. Brides! An Atg8 ubiquitin-like domain (Pfam accession number: PF02991) is of utilitarianism present within MroAtg8 (amino acid positions 16–121, Fig.

3a ). The MroAtg8 and human MAP1LC3B (HsaMAP1LC3B) share 72 % similarity, and their structural superimposition indicates a similar secondary structure, including at the binding sites for Atg7 and brides tubulin, as shown in HsaMAP1LC3B (Fig. 3b ). The amino acid residues which are responsible for that the interaction between Atg8/MAP1LC3B and other autophagy receptors [ 32 ], and the amino acid residues which are important for the formation of two binding pockets are shown in brides, Fig. 3a . We could observe that those important residues were also conserved in crustacean Atg8. Definition Of Utilitarianism! Therefore, the basic residues important for brides electrostatic interaction of MroAtg8 with other proteins could be predicted, possibly including R 11 , R 12 , K 31 , K 50 , K 52 , and R 71 (Fig. 3a ). Spirited Away Creator! Likewise, the picture, residues required for don quixote squire the formation of picture brides two binding pockets in MroAtg8 could be predicted (D 20 , I 24 , P 33 , I 35 , K 52 , L 54 , and F 109 for the formation of the definition, first pocket and I 36 , F 53 , V 55 , P 56 , V 59 , L 64 , I 67 , I 68 , and R 71 for brides the formation of the second pocket) and also mapped in the predicted MroAtg8 tertiary structure (yellow and definition pink areas, respectively; Fig. 3c ), assembling the picture brides, appearance of two adjacent pockets. Identification of an M. rosenbergii Atg8. Squire! a A schematic annotation of M. rosenbergii Atg8 (MroAtg8) and the alignment of crustacean Atg8 proteins with Atg8/LC3 of other species from different phyla. Black shading indicates conserved amino acids while grey shading indicates similar amino acids. Blue boxes indicate the basic residues which are previously known to be critical for brides electrostatic interaction of LC3 with other proteins. Yellow and essays pink bars underneath the alignment indicate amino acids constituting two hydrophobic pockets of picture HsaLC3 (each color represents each pocket) [ 32 ]. Mro, Macrobrachium rosenbergii ; Mni, Macrobrachium nipponense ; Lva, Litopenaeus vannamei ; Sol, Scylla olivacea ; Esi, Eriocheir sinensis ; Dpu, Daphnia pulex ; Dme, Drosophila melanogaster ; Cel, Caenorhabditis elegans ; Dre, Danio rerio ; Xtr, Xenopus tropicalis ; Mmu, Mus musculus ; Hsa, Homo sapiens . Flies Chapter! b Predicted tertiary structures of picture brides HsaLC3 ( a ) and MroAtg8 ( b ) and definition of utilitarianism superimposition of picture brides HsaLC3 and don quixote squire MroAtg8 ( c ). The binding sites for Atg7 (red; amino acid positions 38, 82, and 114) and tubulin (green; amino acid positions 8, 17, 21, 22, 24, 25) in HsaMAP1LC3B are indicated. c Predicted MroAtg8 tertiary structure with hydrophobic surface.

Residues which were predicted to brides form two hydrophobic pockets in MroAtg8 are colored in yellow and pink. Identification of an M. rosenbergii p62. a A schematic annotation of M. rosenbergii p62 (Mrop62) and the sequence alignment of crustacean p62 proteins with p62 of other species from spirited creator different phyla. Black shading indicates conserved amino acids while grey shading indicates similar amino acids. Mro, Macrobrachium rosenbergii ; Ple, Pontastacus leptodactylus ; Dpu, Daphnia pulex ; Dre, Danio rerio ; Xtr, Xenopus tropicalis ; Mmu, Mus musculus ; Hsa, Homo sapiens ; Ame, Apis mellifera ; Spu, Strongylocentrotus purpuratus ; Gga, Gallus gallus ; Cfa, Canis familiaris . b Local alignment of LIR domain sequences of predicted Mrop62 and p62 of other species. Brides! c Predicted tertiary structure of of utilitarianism Mrop62. The predicted Phox and Bem1p (PB1; amino acid positions 6–96), ubiquitin-associated (UBA; amino acid positions 588–634), and LC3-interacting region (LIR; amino acid positions 534–555) domains of Mrop62 are colored in yellow, purple, and red, respectively. Identification of an picture brides, M. rosenbergii Lamp-1. A schematic annotation of M. rosenbergii Lamp-1 and the alignment of crustacean Lamp-1 proteins with Lamp-1 of other species from different phyla are presented. A signal peptide (blue), a mature peptide (green), a conserved transmembrane domain of the epidermal growth factor receptor (TM-EGFR) (yellow), and spirited away cysteine residues are indicated. Black shading indicates conserved amino acids while grey shading indicates similar amino acids. Mro, Macrobrachium rosenbergii ; Mni, Macrobrachium nipponense ; Ple, Pontastacus leptodactylus ; Dpu, Daphnia pulex ; Dme, Drosophila melanogaster ; Cgi, Crassostrea gigas ; Dre, Danio rerio ; Xle, Xenopus laevis ; Rno, Rattus norvegicus ; Hsa, Homo sapiens.

Spatial expression of autophagy markers. Validation of the autophagy marker proteins in female M. Brides! rosenbergii tissues. Western blotting shows the ethnocentrism that, expression of Atg6/BECN1, Atg8/LC3, and p62/SQSTM1 in various tissues, including the hepatopancreas (Hp), ovary (Ov), muscle (Ms), brain (Br), eyestalk (Es), and thoracic ganglia (Tg). The expression of these marker proteins in human squamous carcinoma cells (A431 cell line) is used as a positive control. The amount of total protein loaded per lane was 30 ?g. Immunoperoxidase staining of Atg marker proteins in picture brides, the ovary of M. rosenbergii . Late vitellogenic oocyte (Oc4) with HE stain ( A ). Immunolocalization of Atg6/BECN1 ( B ), Atg8/LC3 ( C ), p62/SQSTM1 ( D ), and Lamp-1 ( E ) in that, the Oc4 of ovarian sections. The insets ( b – e ) show images at a higher magnification where positive staining is picture brides visible as red spots (arrows). F Negative control. Scale bars: 25 ?m ( A and F ) and 10 ?m ( B – E ). Informative Essays! (N, nucleus; Yg, yolk granules; Li, lipid droplets; Mb, cell membrane)

Autophagy refers to picture brides a lysosome-driven process for the degradation of cellular components that are redundant, aged, or damaged [ 37 ]. The control and execution of definition autophagy have been well characterized in the yeast S. cerevisiae [ 38 ] and picture brides the worm C. elegans [ 39 ], and examples are being investigated in human, mouse, the picture brides, fruit fly ( Drosophila melanogaster ), and plants. Statistic Project Examples! Here we present the brides, first study showing that the key genes and proteins involved in the regulation and execution of autophagy are present in crustaceans. All Atg proteins investigated could be identified from M. rosenbergii transcriptomes (see Table 1 ), confirming their widespread conservation amongst animal phyla. It is likely that the autophagic process in ethnocentrism is the belief, crustaceans follows the same pathway as in picture, other animals, engaging a similar set of proteins from the informative, initial triggering step (the activation of the ULK1 protein kinase complex) until the formation of active autophagic vesicles (the autolysosomes). The same hit transcripts were observed for picture brides GABARAP and informative essays GABARAPL1, and ULK-1 and ?2 transcripts. In the case of GABARAP and GABARAPL1, it has been shown that there are at least three human GABARAPLs (1–3) genes which are homologues of the GABARAP gene [ 40 ]. Therefore, GABARAP and GABAAPL1 share a high sequence similarity and both, as well as other GABARAPLs, are categorized within the GABARAP family [ 41 ]. The result from our transcriptome analysis suggested that only a single transcript encoding MroGABARAP exists in the prawn, possibly because other homologous genes may have been lost during evolution. Atg6 (Vps30 in yeast) was initially isolated as an interactor of the anti-apoptotic protein Bcl-2, and picture brides thereafter named Beclin 1 [ 42 ]. Atg6/BECN1 was next shown to statistic project be an essential activator of Vps34 for the initiation of the autophagic process [ 43 ]. High conservation of protein amino acid sequence, especially at the C-terminal region, could be observed in this protein among vertebrates and invertebrates, indicating that those conserved motifs are significant for the activity of picture this protein. In particular, amino acid conservation within the BH3 domain region and of utilitarianism structural superimposition of human BECN1 and picture brides M. rosenbergii Atg6 indicates that M. Examples! rosenbergii and other crustacean Atg6 may interact with Bcl-2, and thereby play a regulatory role in brides, apoptosis.

Upon interaction with BECN1, the informative essays, PI3K class III protein (PI3KC3 in human or Vps34 in yeast) catalyzes the formation of PI3P, which is essential for the recruitment of picture brides autophagosomal membranes [ 44 , 45 ]. Definition Of Utilitarianism! Vps34 protein is the most evolutionarily conserved protein within the PI3 kinase family [ 46 , 47 ]. We did consistently find that crustacean Vps34 showed high conservation in their amino acid sequences with those of other invertebrates and picture brides vertebrates, especially at the domains described as critical for function. Lord Of The Chapter! In fact, the Vps34 protein contains three major domains, namely the C2, PI3Ka, and PI3Kc domains [ 47 ], which were present in the deduced MroVps34. Vps34/PI3KC3 is known to interact with other autophagic proteins such as Atg14 and UV radiation resistance-associated gene (UVRAG) [ 48 , 49 ]. Since Atg14 and UVRAG proteins were found in M. rosenbergii transcriptomes (Table 1 ) and the predicted structural conformation of M. rosenbergii Vps34 is similar to picture human Vps34 (Fig. 2b ), we suggest that MroVps34 may exhibit a similar catalytic activity and bind to Atg14 and UVRAG. Atg8/MAP1LC3B is a protein that is spirited inserted into the nascent autophagosomal membranes upon post-translational lipidation of the microtubule-associated precursor [ 50 ]. Brides! Both length and of utilitarianism amino acid composition were found to brides be similar among representative Atg8/MAP1LC3 sequences from different animal species (Fig. 3 ). This suggested that this protein is don quixote highly conserved among crustaceans and, moreover, among various animals from different phyla. Picture! Consistent with this, the ethnocentrism, predicted structural conformation of brides M. rosenbergii Atg8 exhibits a similar secondary structure to that of human MAP1LC3B, including within the don quixote, regions that are known to be important for picture binding with Atg7 and tubulin proteins [ 51 , 52 , 53 , 54 ]. Besides, Atg8/MAP1LC3B normally interacts with other crucial autophagic receptors, for instance, Atg19, p62, and Atg32, by using two specific hydrophobic pockets, called W- and L-sites (since these two pockets interact with conserved tryptophan and leucine residues within the LIR domain [ 32 ]). We have shown that the don quixote squire, amino acid residues known to be important for MAP1LC3B interaction with other proteins are preserved in crustacean Atg8, suggesting that they are critical for picture Atg8/MAP1LC3B functioning not only in squire, vertebrates but also in picture brides, crustaceans. p62/SQSTM1 is a ubiquitin-binding protein that bridges the autophagy substrate to LC3 through its LIR domain [ 55 ]. Three distinct domains significant for definition p62 activity include a PB1 domain located at the N-terminus that is responsible for p62 self-assembly [ 56 , 57 ]; a UBA domain located at the C-terminus that is important for p62-ubiquitin interacting [ 33 , 58 ]; and a LIR domain that provides an picture, interaction site for binding with Atg8/MAP1LC3B [ 55 ]. We found that p62 in crustaceans are not well conserved with other vertebrate p62, both in their length and amino acid composition. A large amino acid insertion could be observed within the middle region of the crustacean p62 when compared to vertebrate p62 sequences. In regard to the LIR motif, this region is highly conserved among vertebrate species but less conservation could be observed in invertebrate p62 proteins (Fig.

4b ). However, we speculate that the important amino acids ‘WXXL’ (where X is any amino acid) within LIR, which binds directly to MAP1LC3B in mammals [ 32 ], are still preserved in invertebrate p62, including M. rosenbergii , with an exception for the leucine residue which is substituted by other aliphatic amino acids such as valine or isoleucine. Therefore, it is possible that invertebrate p62 may still have binding activity to MAP1LC3B similar to vertebrate p62. Lamp-1 is an don quixote, integral membrane protein of the endosomes and lysosomes, which is involved in the fusion of these organelles with autophagosomal vacuoles [ 31 ]. Picture! Despite low similarity in amino acid composition, we found that the MroLamp-1 preserves basic characteristics of Lamp-1 [ 59 ], including a conserved luminal Lamp domain (namely, the C-terminal TM-EGFR domain that extends to a short cytoplasmic tail). While other vertebrate Lamp-1 contains eight cysteine residues within the luminal region, hence forming four disulfide bonds [ 59 ], MroLamp-1 luminal domain contains only definition four cysteine residues potentially forming two disulfide bonds, and therefore likely not showing the looped-luminal domain with free-moving hinge as present in vertebrate Lamp-1 [ 59 ]. Brides! Considering a cytoplasmic tail of don quixote Lamp-1, this region contains the picture brides, tyrosine-based motif (Y-X-X-I, where X is examples a polar residue) important for brides targeting Lamp-1 to lysosomes [ 60 , 61 , 62 ]. We found that all predicted crustacean Lamp-1 display that significant motif, though their last residues were substituted by valine. Although the intracellular targeting of Lamp-1 was conserved among crustaceans and chapter other animals, a variable amino acid sequence within the N-terminal part and distinct Lamp domain characteristics suggests a different functional activity of crustacean Lamp-1 from those of vertebrates. Based on brides sequence alignment, we found that the sequences of M. rosenbergii and of the flies chapter human proteins at the regions used for producing anti-BECN1, anti-LC3, anti-p62 and anti-Lamp-1 antibodies shared 58.82, 42.86, 41.18 and picture brides 23.37 % identity, respectively, suggesting that antibodies against the human autophagy proteins could be used to detect autophagic proteins in M. Of Utilitarianism! rosenbergii . Picture Brides! We therefore employed immunoblotting and statistic examples immunocytochemistry techniques to brides validate the expression and subcellular localization of Atg marker proteins in M. rosenbergii tissues. Autophagy has been shown to of utilitarianism be present in the epithelial cells of the hepatopancreas and/or intestine in brides, different crustacean species, including Astacus astacus , Eubranchipus grubii and Neocaridina heteropoda , in which autophagic activity was suggested to be essential for away creator digestive cell survival (by eliminating degenerated organelles such as mitochondria) and a proper function of the digestive organs [ 16 , 18 , 19 ]. In our study, we found expression of Atg6/BECN1, Atg8/LC3, p62/SQSTM1 proteins to be widespread in brides, various tissues of M. Of The Flies Chapter 8 Summary! rosenbergii that were investigated (Fig. Brides! 6 ). Regarding Atg8/LC3 expression, there were three immunoreactive bands detected in ethnocentrism is the that, prawn tissues. The largest (18 kDa) and smallest (16 kDa) bands may be considered as the picture brides, LC3-I and-II forms, as compared with the lord flies chapter 8 summary, LC3 expression in the human cell sample (Fig.

6 ). Picture Brides! The middle band, which is present at an apparent molecular weight of 17.5 kDa in some tissue samples, probably represents the intermediate LC3-I that precedes the away, lapidated form of picture brides 16 kDa LC3-II [ 63 ]. Therefore, we suggest that the autophagic process is general to various prawn tissues, although the specific function of informative essays this process within different tissues still requires further investigation. In the ovary, we could observe that the picture, levels of Atg6/BECN1, Atg8/LC3, p62/SQSTM1 and Lamp-1 expression within the cytoplasm increased in correlation with oocyte maturation, while no immunoreactivity was found in project examples, the follicular cells. We hypothesize that the autophagy process may be essential for reproductive activity in crustaceans. However, whether autophagy is picture brides a part of cellular controls that regulate oocyte differentiation requires further investigation. The present data supports the of utilitarianism, high evolutionary conservation of autophagic components in the crustacean lineage, as implicated by the existence of brides several key autophagy-related genes and proteins, and a high degree of sequence conservation of crucial autophagy marker proteins among crustaceans and other representative species from different phyla. This reflects a selection pressure for autophagic process during crustacean evolution. In addition, we have demonstrated that antibodies raised against human autophagy marker proteins can be used for monitoring the autophagic process in a crustacean model. Learning how autophagy is regulated in the giant freshwater prawn could open up new avenues for novel management in limiting infections, organ stress, as well as nutritional and metabolic imbalances due to squire feeding and starvation, which can ultimately impact on crustacean aquaculture. Adult females M. rosenbergii were obtained from brides a local commercial farm in Ayutthaya province, Thailand.

The prawns were anesthetized by immersion in ice water, and then the spirited creator, ovaries, hepatopancreases, muscles, brains, eyestalks, and thoracic ganglia were dissected out. Fresh tissues were kept at picture brides ?80 °C for further use in transcriptome production and Western blotting (described below). For histological and immunohistochemical studies, tissues were fixed in ice-cold 4 % paraformaldehyde for 24 h. Don Quixote Squire! After washing with 70 % ethanol five times, specimens were then dehydrated in increasing concentrations of picture ethanol, cleared in xylene and infiltrated with paraffin, using an automated tissue processor. The paraffin-embedded ovarian tissue blocks were cut at 5 ?m thickness and prepared for don quixote squire immunofluorescence staining. Transcriptome production and de novo assemblies of CNS and ovary of M. rosenbergii. Transcriptome data of M. rosenbergii was obtained from a previous study [ 26 ]. For transcriptome production, in brief, total RNA was isolated from the CNS (inclusive of the eyestalk, brain, thoracic ganglia, and abdominal ganglia) and the ovarian tissues using Tripure isolation reagent (Roche, IN, USA). The quality of total RNA of picture each tissue was checked by gel electrophoresis and spectrophotometry (NanoDrop 1000; Thermo Fisher Scientific, DE, USA). Twenty micrograms of informative total RNA of each tissue was sent to BGI, Hong Kong, for library construction. Briefly, complementary DNA (cDNA) was synthesized from picture brides purified and fragmented RNA samples using BGI standard workflow for de novo RNA-seq transcriptomes ( http://bgiamericas.com/ ). Ethnocentrism Is The Belief! The cDNA libraries were constructed based on picture brides polymerase chain reaction (PCR) amplification using random hexamer-primed cDNAs. Then the samples were sequenced using Illumina HiSeq 2000 instrument (Illumina Inc.) and only clean reads were used for further sequence assembly. De novo assembly was generated by project, SOAPdenovo software [ 64 ] using a combined CNS and ovary dataset with parameter set as follows: seqType, fq; minimum kmer coverage = 4; minimum contig length of 100 bp; group pair distance = 250.

All sequence data of M. rosenbergii CNS and ovary transcriptomes were deposited in the NCBI database (Accession number: SRP049917). Gene mining, characterization, and sequence alignment. Searching of picture brides Atg proteins in M. Lord Of The Chapter 8 Summary! rosenbergii transcriptome dataset was carried out by using tBLASTn program in CLC Main Workbench Version 6.0 (CLC Bio-Qaigen, AsiaPac, Taiwan) for picture brides which known Atg proteins (from mammals, yeast, and insects) were used as queries. The parameter was set as follows: matrix, BLOSUM62; e-value, 100. To search for transcripts encoding Atg proteins in other crustaceans, we used protein queries of known Atg proteins to search within publicly accessible databases [non-redundant (Nr), expressed sequence tags (ESTs), transcriptome shotgun assembly, and Daphnia genome [ 65 ] databases] by using NCBI tBLASTn program ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ), which was restricted to crustaceans (taxid: 6657) and ethnocentrism belief that the parameter was set as follows: expect threshold = 10; matrix, BLOSUM62; gap costs, existence = 11 and brides extension = 1. Is The! Nucleotide sequences obtained from BLAST searches were translated into amino acid sequences using the CLC Main Workbench Version 6.0 and then primary sequence analyzed manually based on homology to other known proteins. Picture! Protein alignment of definition crustacean Atg proteins with other previously known Atg proteins was performed using MEGA6 [ 66 ] and picture brides GeneDoc [ 67 ] softwares. Amino acid sequences used in the alignments, as well as their accession numbers, are provided in Additional file 2 . All deduced M. rosenbergii Atg proteins were finally used for of the flies 8 summary BLASTP search against the Nr protein database for further sequence confirmation. Picture Brides! The BLAST hit with lowest e-value for each M. Don Quixote! rosenbergii Atg protein is detailed in Additional file 1 . Prediction of protein tertiary structure and critical sites. The tertiary structure of selected Atg proteins were predicted using the online tool Protein Homology/analogY Recognition Engine V 2.0 [ 68 ]. Visualization of predicted protein tertiary structure as a 3 dimensional model, illustration of hydrophobicity, superimposition of proteins, and structure labeling were performed using the UCSF Chimera version 1.8.1 [ 69 ]. The specific domains and critical sites were predicted by InterPro [ 70 ]. Primary antibodies used in western blotting and immunohistochemistry.

Rabbit polyclonal antibody against BECN1 (Sigma-Aldrich, USA) was produced using synthetic peptide corresponding to picture amino acid positions 329–345 of human BECN1 as immunogen. Rabbit polyclonal antibody against LC3B (Sigma-Aldrich, USA) was raised against the human LC3B at amino acid positions 2–15. Rabbit polyclonal antibody against definition, p62 (Santa Cruz, Biotechnology, USA) was produced against the human SQSTM1/p62 at amino acid positions 375–425. Mouse monoclonal antibody against Lamp-1 (BD Science) was produced against human Lamp-1 at the amino acid positions 25–224. Western blot analysis of Atg8/LC3, Atg6/BECN1, and p62/SQSTM1. Fresh tissues were homogenized and sonicated in brides, a lysis buffer (0.2 % NaDoc, 1 mM Na 3 VO 4 and 50 mM NaF) and then centrifuged at examples 10,000? g , 4 °C, for 1 h to brides obtain tissue extracts.

The supernatants were collected and protein concentrations measured with the Bradford reagent (Sigma). For a positive control, human squamous carcinoma cells (A431 cell line) were sonicated in is the that, lysis buffer and protein concentrations measured. Brides! Subsequently, 30 ?g of total proteins were separated on a 15 % SDS-PAGE gel and transferred onto a polyvinylidene fluoride membrane (GE Healthcare Life Sciences). Nonspecific binding sites were blocked with a blocking buffer [5 % non-fat dry milk in 0.2 % Tween 20 in ethnocentrism is the belief that, 0.1 M phosphate-buffered saline (PBS), pH 7.4] for brides 2 h at room temperature. The membranes were incubated overnight at 4 °C with the following primary antibodies: either rabbit anti-LC3 (dilution 1:500), rabbit anti-p62/SQSTM1 (dilution 1:200), or rabbit anti-BECN1 (dilution 1:500). The membranes were washed three times for 5 min each with washing buffer (0.01 % Tween 20 in 0.1 M PBS) and then incubated for 2 h at squire room temperature in the peroxidase-conjugated secondary antibody [goat anti-rabbit IgG-HRP (SouthernBiotech) dilution 1:5000]. Brides! Antigen-antibody binding was detected using an spirited away, enhanced chemiluminescent substrate (Thermo Scientific) and exposed to Amersham Hyperfilm ECL (GE Healthcare Life Sciences). Immunoperoxidase staining of Atg proteins in brides, the ovary of M. rosenbergii. Immunoperoxidase staining was carried out following a previously described protocol [ 71 ] with some modifications.

Briefly, ovarian sections were sequentially deparaffinized and rehydrated in xylene and decreasing concentrations of ethanol. Endogenous peroxidase was blocked by immersing sections in 3 % H 2 O 2 in informative essays, methanol for 30 min. Subsequently, the picture, sections were immersed in 1 % glycine in 0.1 M PBS to squire block free aldehyde groups and then incubated in a blocking serum (10 % bovine serum albumin in 0.1 M PBS, pH 7.4) for 2 h at 4 °C to block non-specific bindings. Picture Brides! Sections were incubated overnight at 4 °C with either rabbit anti-LC3 (dilution 1:500), mouse monoclonal anti-Lamp-1 (dilution 1:500), rabbit polyclonal anti-p62/SQSTM1 (dilution 1:500), or rabbit polyclonal antibody anti-BECN1 (dilution 1:500), in blocking serum. Definition! After washing with PBS, sections were incubated in picture brides, secondary antibody, either goat anti-rabbit IgG-HRP (Abcam) or goat anti-mouse IgG-HRP (SouthenBiotech) at the dilution 1:500 in blocking serum, for 2 h at room temperature. Negative controls were performed by omitting the primary antibodies. The color was developed with an AEC substrate kit (Invitrogen). Ethnocentrism Belief! Finally, sections were observed and photographed under the Nikon E600 light microscope. BECN1, Beclin 1; Lamp-1, lysosomal-associated membrane protein 1Vps, vacuolar protein sorting; MAP1LC3, microtubule-associated proteins 1A/1B light chain 3; SQSTM1, sequestosome. This research was supported by a grant from the Office of Higher Education Commission (OHEC) and Mahidol University (MU) under the National Research University Initiatives, an OHEC-MU and brides the Thailand Research Fund (TRF) Distinguished Research Professor Grant to Professor Prasert Sobhon, a TRF-RGJ Ph.D. Examples! Scholarships to brides Saowaros Suwansa-ard and is the belief Wilairat Kankuan; and an ARC grant to Dr.

Scott Cummins. We also thank the financial support from the Consorzio InterUniversitario per le Biotecnologie (Trieste, Italy) and from the Associazione per la Ricerca Medica-Ippocrate-Rhazi (Novara, Italy) to Miss Wilairat Kankuan. This work was supported by a grant from the Office of Higher Education Commission (OHEC) and Mahidol University (MU) under the National Research University Initiatives, an OHEC-MU and picture brides the Thailand Research Fund (TRF) Distinguished Research Professor Grant to PS. SS was supported by a TRF-RGJ Ph.D. Scholarships. WK was supported by a TRF-RGJ Ph.D. Scholarship, the Consorzio InterUniversitario per le Biotecnologie (Trieste, Italy), and the Associazione per la Ricerca Medica-Ippocrate-Rhazi (Novara, Italy). SFC was supported by an ARC grant. Availability of data and materials. The datasets supporting the conclusions of this article are available in the NCBI Sequence Read Archive (SRA) depository (SRA Accession number: SRP049917; http://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP049917 ). Additional supporting data are included as additional files. SS performed the bioinformatics analyses and wrote the manuscript.

WK performed the project, Western blot analysis and immunohistochemistry, and brides wrote the manuscript. TT, JS, NK, TK and KK contributed the transcriptomic data and analysis. SFC, CI, and don quixote squire PS provided conception, laboratory support, critical revision of the brides, manuscript, and don quixote squire final approval of the version to be published. The authors have declared that no competing interests exist. Ethics approval and consent to participate. Ethics is not required for the study. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to picture brides the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated. Saowaros Suwansa-ard 1 4 Wilairat Kankuan 1 5 Tipsuda Thongbuakaew 1 7 Jirawat Saetan 2 Napamanee Kornthong 3 Thanapong Kruangkum 1 Kanjana Khornchatri 1 Scott F. Cummins 4 Ciro Isidoro 5 Email author Prasert Sobhon 1 6 Email authorView author's OrcID profile 1. Department of Anatomy, Faculty of Science Mahidol University Bangkok Thailand 2. Department of Anatomy, Faculty of examples Science Prince of picture Songkla University Hatyai Thailand 3. Chulabhorn International College of Medicine Thammsat University Phathumthani Thailand 4. Faculty of Science, Health, Education and ethnocentrism is the Engineering University of the Sunshine Coast Maroochydore Australia 5. Laboratory of picture brides Molecular Pathology, Department of Health Sciences Amedeo Avogadro University Novara Italy 6. Faculty of Allied Health Sciences Burapha University Chonburi Thailand 7. School of Medicine Walailak University Nakhon Si Thammarat Thailand. .RIS Papers Reference Manager RefWorks Zotero. .BIB BibTeX JabRef Mendeley.

.RIS Papers Reference Manager RefWorks Zotero. .BIB BibTeX JabRef Mendeley. Over 10 million scientific documents at your fingertips. 2017 Springer International Publishing AG. Part of Springer Nature.

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100 Easy Argumentative Essay Topic Ideas with Research Links and picture brides, Sample Essays. VirginiaLynne has been a University English instructor for over 20 years. She specializes in helping people write essays faster and easier. Need a great argument topic? Below I give over 100 ideas. Ethnocentrism. You'll write faster and easier if you pick a topic based on: Knowledge: Picking a topic you already know a lot about can make research faster and picture brides, easier. Interest: Picking a question you want to know more about can make this paper more interesting.

Available Sources: I give links to many sources. Check those for articles first and if you find some, your work is half done. I also save you time by giving you links to videos and sample student essays. Check out my guides for writing papers too. Good luck! If you get a good grade, be sure to lord 8 summary come back and tell me! 1. Instructions for how to (and how not to) pick a topic. 2. Lists of picture topic ideas (in the don quixote squire categories of brides food and health, obesity and dieting, recycling and the environment, families and relationships, and of utilitarianism, science and technology, with videos and many links to research and picture, student essay examples. 3. Step-by-step instructions for how to definition write your essay.

Choosing a topic that everyone is brides, talking about makes writing an argument essay easier. Make sure you choose a question that doesn't have an project examples answer people already agree on. Picture Brides. Pick a reader that doesn't agree with you, so that you are not preaching to the choir. It also helps if the flies chapter topic is something everyone has an opinion about: this will make it easier to get examples to picture back up your essay, either from ethnocentrism is the articles or from people you interview. Finally, you probably want to brides pick a topic that is don quixote, interesting to you and picture brides, that you care about. Steer clear of overused topics like abortion, gun control, and the death penalty. For one thing, your instructor has already read far too many of these essays and informative, is not only probably bored with the topic, but also has already heard everything you might say. Moreover, although those may seem like easy topics, they really aren't, because most people are set in picture brides their ways about don quixote squire, these issues and it is brides, hard to of the think of an picture brides argument that might change their minds. Is Deforestation Worth it?

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Diet Topic Articles from Psychology Today . Flies. What can we do to help people around the world have clean water to drink? Is hunting good or bad for the environment? Should the horns of picture wild rhinos be removed to prevent them from being poached? Can we protect wild areas and animals by promoting eco-tourism? Should the government discourage or regulate oil drilling in the gulf of Mexico? What are the ethnocentrism is the belief that dangers of fracking? Does recycling really make a difference? Should all states adopt a deposit on soft drink bottles and cans in picture brides order to definition of utilitarianism promote recycling? Should schools require students to bring refillable containers for brides, water and statistic project examples, other beverages rather than disposable ones?

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Are landfills a good idea? Where can we use solar, wind, and other alternative energy sources effectively? What is the best way to encourage alternative energy use? (Government regulations? Incentives? Helping companies that produce these products? Advertising?) The World Bank on Environment: Information and research about environmental issues around the world. The U.S. Picture Brides. Environmental Protection Agency: On the science and technology page, find research and information under different topics like water, pesticides, or ecosystems.

Discover Magazine: Search the environmental topics page for ethnocentrism belief that, your topic. U.S. Government Recycling and Conservation: Statistics and information. Should mothers stay home with their children? Do long distance relationships work?

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How does birth order affect children? Do older people make better parents? Have cell phones and social media made families closer or not? How should (or shouldn't) you use social media in a dating relationship? How long should people date before they become engaged? What makes people have a happy, long-lasting marriage? Are the expectations raised by romantic movies damaging to real relationships? Are Beauty Pageants Good for Kids? What is picture, nanotechnology and how has it already changed our lives?

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Is there a balance between quantity and quality of life? How can 3-D printers be used effectively? SciTech Daily: Science and picture, new technology news and research reports. MIT Technology Review: Massachusetts Institute of statistic Technology's website for explaining new technologies. If you want to write a quick and easy argument paper, follow these simple steps: Pick a topic question from the lists above. Decide your answer to the question (this is picture brides, your beginning thesis). Write down everything you know about the topic. Of Utilitarianism. Talk to your friends or family to find out what they know, have heard, or have read recently about the topic (have them give you the source if they know it). Look at some of the research articles or web sites about that topic. Look back at your question and brides, refine your answer. After gathering information, you may want to change it.

Write down three or more best reasons for your answer (these are your topic ideas for the body of your essay). Using those reasons, look at the articles you've read or the statistic project ideas you've already written down for some evidence to support those reasons (this is the brides backup evidence for each topic sentence). Don Quixote Squire. Write your outline, then follow it to write your paper. Do you have to do research for your paper? Easy Argumentative Essay Topics for College Students. by Virginia Kearney 5. Funny Argumentative Essay Topic Ideas. by Virginia Kearney 8. 100 Current Events Research Paper Topics with Research Links. by Virginia Kearney 16. How to Write a Proposal Essay/Paper. by Laura Writes 40. 100 Argument or Position Essay Topics with Sample Essays. by Virginia Kearney 37. 100 Science Topics for Research Papers. by Virginia Kearney 108.

This is a great resource. I've just entered into college, and picture brides, didn't know where to begin writing my first argumentative essay. Thanks so much - voted up :) Do you know or have you written of anything to do with the argumentative essay of chapter should smoking be banned because I have to do essays with research and I need some reliable sites. Virginia Kearney 3 weeks ago from United States. Hi Aashi! I'm glad that younger students are finding my work too. Although I now teach college students, I started my career teaching in your grade for several years. I will have to put together some topics for primary grade students. Until then, you might want to look at my High School Topics, which have many ideas which are good for your age too.

I am in primary school in grade 6 and I want some good topics for primary students. Good job making this page. Picture. I don't know what to do at first. Creator. I was clueless and was browsing for answers but none of them made sense except for brides, this. Thank you very much! I think the ideas are wonderful and project examples, are very helpful! I am a Junior in high school and I have to write an brides argumentative paper. Informative. Your insight on how to do so has been extremely helpful. I wanted to brides thank you for your intelligence on how to write an argumentative paper. Thanks! This website was very useful for picking out project examples, a topic for my essay.

Again, thank you for helping me out! King of Stuff 8 months ago. I find this website very interesting and helpful. Thank you for picture, making it! Your tips on writing essays is really helping me out. Mr. fluffypants 8 months ago. I love this article. Ethnocentrism. You have helped me with my school essay. Thank you! letter pile 8 months ago.

I LOVE this website. Thank you so much for writing it! It has helped me so much! Virginia Kearney 8 months ago from United States. Picture Brides. Hi Nataly! You are welcome to shift the focus of the of the flies chapter questions to whether governments should take action. My questions and topic ideas are just a starting point. I teach my students that there are a variety of ways to solve problems and one of brides those is by informative essays, having governments or larger groups take action. However, I want my students to focus more on how they and their audience can personally take responsibility and brides, action, so often my questions are more locally written. In my class, I focus on having my students address a very particular audience in their persuasive essays because effective arguments come from really thinking carefully about the statistic viewpoint of the other person and developing points that would persuade that person.

In my class, I do allow TedTalks as sources if the student is using other sources as well. However, I don't think all professors do accept that type of source. Picture Brides. Most people who do TedTalks have written out their arguments in ethnocentrism is the belief papers or books, so I'd suggest you research to brides see if there is an online paper you can cite as well. Squire. I find your lists great and really appreciate the idea of picture brides providing useful links. Belief That. Still, I would rather shift the focus of some questions so that they ask students to think more globally, from the point of view of the state and the society.

For example, should the state take actions to brides prevent high rate of divorces, which ones? Are people in developed states responsible for providing water and chapter, food to the starving people around the globe?, etc. I also wanted to ask you if TedTalks videos are officially recognized as credible resources. Have you ever heard of brides any cases when professors forbid to use it? Thank you for a good work! Hulya Gulyurt 9 months ago. This helped me so much with my homework, thank you! Great Efforts . Ethnocentrism Belief. Well done. Picture. should guns be allowed on school campus. Virginia Kearney 13 months ago from United States. This is an interesting topic idea bojoi--and definitely one that would be controversial.

I'd love to project examples hear how you would develop your thesis. preetyradd 14 months ago. i think this is cool i got a good grade on my essay thanks. Kanwal asif 14 months ago. Thank u so much God bless u. Picture. Virginia Kearney 17 months ago from United States. Of Utilitarianism. Glad this will help you three keys! ThreeKeys 17 months ago from Australia. Im about to try out your suggestions in this great article. Im excited to see what the outcome will be in how I take a more pointed or comprehensive approach in a written debate so to speak. Thanks so much! Thank you it is brides, really helpful.

Thank you so much for the topics. Trisha Roberts 3 years ago from Rensselaer, New York. Love the great ideas! Absolutely love the list you shared with us. Thank you so much for definition, this Article! Kalai 3 years ago from Petaling Jaya, Malaysia. Sometimes i find that the picture most easy or obvious topic the hardest to argue about. Away. The less the words the greater the headache. When we prepare for debates, each word has the ability to make or break the case.

Rae Saylor 3 years ago from Australia. What an brides interesting range of definition of utilitarianism ideas and tips! Massive thanks for writing this, pal! Voted up :) Eiddwen 3 years ago from Wales. A great hub Victoria ;thanks for sharing and I vote up. Dianna Mendez 3 years ago. Picture Brides. This is very useful to squire those who must teach essay writing (and to picture those who must write them). I know I will be using this next time I teach English Comp.

Voted up++ ExpectGreatThings 3 years ago from Illinois. Wow! This is a very impressive list and great instructions. I like how you were able to definition write the questions without giving away your position on each topic. - Ginger. Eric Dierker 3 years ago from Spring Valley, CA. U.S.A. Picture. Very interesting. Fun ideas and great food for thought. Copyright 2017 HubPages Inc. and squire, respective owners. Other product and company names shown may be trademarks of their respective owners.

HubPages ® is a registered Service Mark of HubPages, Inc. HubPages and Hubbers (authors) may earn revenue on this page based on affiliate relationships and advertisements with partners including Amazon, Google, and picture brides, others. Copyright 2017 HubPages Inc. and respective owners.

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with the media but at the same time realizes that some things fall under the 1st amendment. In this essay I will discuss many parts of the media and some of its past. I will go into the history of the media , the role of television, political campaigns and the media , government and the media , regulation. The Medium of Culture and the Impact of Media. Culture and the Impact of Media Abstract In recent years, media has had the largest impact on belief culture in picture, society. There are many different types of medium; these different types allow people to spread ideas quicker and to a larger audience. Observations made through media influence people in of the flies chapter 8 summary, different. Statement of Central Idea (Chp.

11) • • List the brides, three components of the statements of central idea Write an accurate statement of central idea for a speech that lacks such a statement Speaker Credibility (Chp. 11) • • • Define speaker credibility Identify and statistic, define the two dimensions of speaker. New Age Myth and the Role of the brides, Media: a Study of Dr Mahathir Mohamed. understanding of ourselves and statistic examples, our culture is based largely upon what we are told by the media . Yet much of the media’s content includes unexamined assumptions and myths, an agenda set by those in picture, control of the media . These myths are stories, themes, and ideas that embody an ethnocentrism is the belief, aspect of culture. Politicians. ?UOP COM 220 Week 1 Assignment Bias, Rhetorical Devices, and Argumentation. Week 1 Assignment Bias , Rhetorical Devices, and brides, Argumentation Check this A+ tutorial guideline at flies 8 summary, http://www.assignmentcloud.com/com-220/com-220-week-1-assignment- bias -rhetorical-devices-and-argumentation For more classes visit http://www.assignmentcloud.com Assignment: Bias , Rhetorical Devices. Analysis of picture brides, Obama Speech in Egypt.

A New Beginning Outline Thesis: Obama used persuasive techniques such as, ethos, logos and pathos to deliver his message of a new start in the relations between the east and the west. I. Ethos A. Spirited Away Creator! Humble man 1. Thanking the Egyptian people B. Picture Brides! Fair 1. Admitted the. journal describes how it is important that parents become aware of the nature of social media sites, given that not all of them are healthy environments for children and adolescents. This journal “The Impact of Social Media on Children, Adolescents, and Families” the writers clearly appeal to parents through. ?The Freedom of Speech or The Freedom of Equality College is a time when most individuals are experiencing major changes and begin to explore new perspectives. The key factors in lord 8 summary, changing the perspective of an individual are the transition in becoming more independent, creating new insights and peer. Society and Media Culture The advancement of brides, technology over the past decade has impacted the lives of examples, individuals and changed our culture within the United States. These advancements have created an entirely new type of brides, culture that is heavily focused on media usage. “ Media culture” has reinforced. that we’re not around, we don’t question the media that is exposed to us; we mainly just go with whatever political party we’re most affiliated with. Although “reporters were trained to keep personal beliefs to themselves when writing about definition events” (“ Media Bias ” 1), reporters are taking advantage of the.

examples using a listening checklist to illustrate what went wrong. 2.- Describe a speech or presentation where speaker volume was a problem (exclude examples of brides, malfunctioning PA systems). How did this experience affect your bias concerning the definition, topic and picture brides, speaker? Why is it important to consider and ensure. Course (UOP) For more course tutorials visit www.com220.com COM 220 Week 1 CheckPoint Research Paper Topic COM 220 Week 1 Assignment Assignment Bias , Rhetorical Devices, and Argumentation COM 220 Week 2 DQ 1 and DQ 2 COM 220 Week 2 Checkpoint Gathering and examples, Evaluating Information COM 220 Week 3. going to continue working on brides your Persuasive Speech . Refer to the instructions below for today’s work day guidelines. Project Goal: The goal of this project is to choose a position, research both sides of the subject matter, and prepare and write a persuasive speech that is geared towards OPPONENTS. Role of Indian Media in the New Age The following is the essays, speech given by picture Shri Kapil Sibal, Minister of HRD and C IT, on the topic: Role of Indian Media in informative, the New Ag, presiding as the Chief Guest for the 'Press Club Awards for Excellence in Journalism' , on 5th May, 2012 at The NSCI, Worli, Mumbai.

?The Freedom of Speech In the United States, citizens have several rights that are protected by the United States Constitution. In the First Amendment to the United States Constitution, the Freedom of Expression ( speech ) is recognized. The Freedom of Speech is the right to express any opinions without. COM 220 Uop Course Tutorial/ Tutorialoutlet. more course tutorials visit www.tutorialoutlet.com COM 220 Week 1 CheckPoint Research Paper Topic (Uop) COM 220 Week 1 Assignment Assignment Bias , Rhetorical Devices, and Argumentation (Uop) COM 220 Week 2 DQ 1 and DQ 2 (Uop) COM 220 Week 2 Checkpoint Gathering and Evaluating Information (Uop) . COM 220 UOP Course Tutorial/ Uoptutorial. COURSE For more course tutorials visit www.uoptutorial.com COM 220 Week 1 CheckPoint Research Paper Topic COM 220 Week 1 Assignment Assignment Bias , Rhetorical Devices, and Argumentation COM 220 Week 2 DQ 1 and DQ 2 COM 220 Week 2 Checkpoint Gathering and Evaluating Information COM 220 Week 3. Paul Mitchell Persuasive Language.

landed in the media for brides appalling acts performed to a developmentally delayed 17-year-old girl. In his article on definition of utilitarianism the issue the 3AW broadcaster and writer, Neil Mitchell, portrays to us his one-sided opinion and disgust towards the teenagers. Mitchell uses an picture, abundance of persuasive techniques to engage.

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coincidence essay Often, the hardest words to write in an essay are the opening ones. When you’re doing the picture, first draft, I’d suggest just writing your way through the introduction without worrying too much – you’ll want to come back to it when you’ve got the body and the conclusion of your essay firmly decided upon. But when you’re revising your essay, you should concentrate on informative, making the picture brides, opening paragraph as strong as possible – the person marking it will inevitably start forming an opinion of of the chapter your essay’s worth within the first few words. If you want to go beyond the standard, rather bland, introduction of picture “In this essay, I am going to…” you might want to try out some of squire these openings for your essay: My favourite way to start off an essay is with a pithy quote. I don’t recommend picking one of the hilarious quotes listed on your Facebook profile, though; instead, try to find something as relevant as possible. If you’re writing an English essay, a good opening might be a line from picture, one of the plays or books that you mention in don quixote the essay – particularly if that line sets up the angle that you’ll be taking on the question. If you’re studying Politics, a quote from a politician can work very well. Again, make it relevant to the essay (if you’re writing about picture brides, Thatcherite Britian, “There’s no such thing as society” would be fine, but less so if you’re writing about the away, Middle Ages…) When you can’t quite find a subject-specific quote that works, a famous proverb or saying (particularly one which might make your tutor smile) could work well.

The best source of quotes are the textbooks you’re using, but if you’re still stumped, try: Another good way to open an essay is to brides define any key terms in the question. Statistic Project Examples? This might seem a bit of a bland opening, but it’ll show your tutor or examiner that you know what you’re talking about picture, – and ethnocentrism, it sets the picture brides, parameters of the discussion. For example, a word like “ideology” can be very loaded: if it appears in the question, you’ll want to make clear what definition you’ll be using throughout your essay. Try not to definition spend too long defining terms, though; whilst an picture brides, indepth discussion of one word can be fruitful, doing this for definition of utilitarianism, every single word in the question will just indicate that you’re not quite sure what your argument is… Famous example – A.D. Nuttall’s A Common Sky: If you’re feeling brave, you could open your essay with a bang by making a bold statement. Picture Brides? (An easy way to definition do this is to completely disagree with the question or prompt that you’ve been given.)

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