Characterizing HP’s from Leptospira Interrogans

Characterizing HP’s from Leptospira Interrogans Introduction: Genome sequencing projects and genetic engineering has revealed many aspects of complex cellular environment containing large number of proteins. Despite sequences of most of organisms are available and proteins coded are studied experimentally, there are some proteins whose functions are unknown, need to be characterised(5). Such proteins are known as Hypothetical proteins (HP) sequences of which are known but there is no evidence of experimental study(6). There is extensive need to study and classify these hypothetical proteins which can open new way to design drug molecules against infectious organisms. Functional annotation of HP’s involved in infection, drug resistance, and essential biosynthetic pathways is important for development of the potent antibacterials against infectious agents. Improved understanding of these proteins may make them potential targets of antimicrobial drugs [26]. Leptospira interrogans is gram negative spirochete, having an internal flagella is p athogenic which causes Leptospirosis (1)(2), other serovars (strains) are distinguished on the basis of cell surface antigens. These are infectious to animals, but through animal urine can be spread to human(3). Leptospira enters in body via broken skin, mucosa and spreads in body, if immune system fails to stop the growth of bacteria it cause severe hepatic and renal dysfunctions(4). This present study highlights thein silicostudies to characterize HP’s from Leptospira interrogans. Methods: Sequence Retrieval: KEGG (Kyoto Encyclopedia of Gene and Genomes) is a large collection of databases having entries of genes, proteins, pathways in metabolism and diseases, drug and ligands of organism (7). We have selected the Sequences of 12 hypothetical proteins of Leptospira interrogans randomly from KEGG database (www.genome.jpg/kegg).   Pfam: Pfam is curated Protein families database, it uses jackhmmer programme (HMMR3). To give profile HMM ( Hiden Markov Model) with PSI-BLAST, which were searched against UniProt(9). However, to include protein in a family its domain and sequence bit scores must be equal or above the Gathering Thresholds (GA). Pfam gives Pfam A families which are manually curated and Pfam B families generated automatically(8). Batch CD search: Hypothetical Protein sequences were searched for conserved domains at batch CD search, which gives results by using MSA and 3D structures for homologous domains available on Pfam and SMART(9)(10). ExPASy-ProtParam tool: ProtParam tool (www.expasy.org/tools/protparam.html) was used to estimate physicochemical parameters of hypothetical proteins(11). Query protein can be submitted in form of SWISS/TrEMBL ID or protein sequence. Server provides directly calculated values of pI/MW (Isoelectric point, Molecular Weight), Percentage of each amino acid, Extinction coefficient (EC), Instability Index (II)(12), Aliphatic Index (AI) and GRAVY (Grand Average of Hydrophobicity). SOSUI server: Amphiphilicity index and Hydropathy index of query protein sequences were calculated by SOSUI server which categorises protein into cytoplasmic or transmembrane nature(13). Protein-Protein Interaction network: Protein in the cell environment interacts with other proteins, in silico these interactions were studied by STRING v9.1 (Search Tool for Retrieval of Interacting Genes). STRING is a large repository of protein-protein interactions involving functional interactions, stable complexes, and regulatory interactions among proteins(14,15). Figure 1. Shows resulting protein-protein interaction network of selected hypothetical proteins, for better understanding interaction networks should be seen on server site. Disulfide-Bonding in Protein Disulfide bonds among cysteine residues in protein plays an important role in folding it into functional and stable conformation. DISULFIND server utilizes SVM binary server to predict bonding state of cysteins, these cysteins are paired by Recursive Neural Network to show disulfide bridges(16). Protein Structure Prediction: Protein structure prediction server (PS)2 (17) requires query sequence in fasta format to generate 3D structure by comparative modelling(18). Server utilizes consensus strategy to find template using PSI-BLAST and IMPALA. Query sequence and template aligned by T-coffee, PSI-BLAST, and IMPALA [13]. 3D structures are predicted from template using MODELLER and visualised by CHIME, Raster3D. Resulting 3D structural model of selected hypothetical proteins are shown in Figure 2. Ligand Binding site Prediction: Q-site finder(19) server was used for binding site prediction in selected proteins. Server uses energy based methods to find clefts on protein surface for ligands(20). These hot spots for ligand binding have predicted after ranking their physicochemical properties as hydrophobicity, desolvation, electrostatic van der waal potentials. Discussion: ProtParam tool computes different physicochemical parameters depending on the queries submitted to the databases. Isoelectric focusing separates proteins according to pI where pH gradients are developed(21). Predicted pI via server may not be adequate because in case of high number of basic amino acids and lower buffer capacity. By using pH gradients and calculated pI, proteins can be separated experimentally. MW of proteins along with pI is used for the 2D gel electrophoresis. EC shows a light absorbed by a protein relative to their composition at a specific wavelength. EC given (Table 1) are calculated with reference to Tryptophan, Cysteine, Tyrosine (11). Instability index (II) refers to the stability of the protein in test tube(22). Among studied proteins giÇâ‚ ¬24214908, giÇâ‚ ¬24215664, giÇâ‚ ¬24216444, giÇâ‚ ¬24213620, gi|24213945 were found to be unstable, and rest are stable (proteins with II above 40 are unstable). Aliphatic amino acid constitute s the aliphatic index (a relative volume of aliphatic side chains). Increased AI results into a hydrophobic interactions and thus gives thermostatic stability to protein, predicted AI and II shows inverse relation for stability except these two proteins giÇâ‚ ¬24215664 and giÇâ‚ ¬24215909. GRAVY(23) values are a ratio of all hydropathy values of amino acids to the number of residues in sequence. Smaller the GRAVY(23) more hydrophilic is protein, giÇâ‚ ¬24214908 and gi|24213945 proteins found the most hydrophilic. In case of 3D structure hydrophilic domains tends to be on exterior surface, while hydrophobic domains avoids external environment and forms internal core of the protein. Search of family for hypothetical proteins based on conserved domains having consensus sequence in their structure is given in Table 3. Hypothetical protein giÇâ‚ ¬24214908 found to be a member of GH18_CFLE_spore_hydrolase, Cortical fragment Lytic Enzyme bearing a catalytic doma in from glycosyl hydrolase, an enzyme used in breaking a spore peptidoglycans so as to activate it for germination when favourable conditions are available. Hypothetical protein giÇâ‚ ¬24215649 from PDZ_serine_protease involved in protein reassembly and work as a heat shock protein. Protein giÇâ‚ ¬24215664 belongs to Leucine-rich Repeats (LRR), ribonuclease inhibitor like family. LRR are motifs having role in protein interactions in complex networks. S-adenosylmethionine decarboxylase (AdoMetDC) enzyme for biosynthesis of spermine and spermidine by decarboxylation of SAM belongs to Ado_Met_dc family (giÇâ‚ ¬24217373). Pilz domain in giÇâ‚ ¬24213620 is found in bacterial cellulose synthase and other proteins that forms biofilm around a bacterium and involve in effluxing drug(24). Hypothetical protein giÇâ‚ ¬294827583 (FecR superfamily) is involved in Iron transport system in bacterial membranes, Fe3+ (insoluble) loaded on citrate carrier is sensed b y FecR protein found in periplasmic space in bacterial membrane(25). Protein sites are predicted as cytoplasmic, host associated, extracellular, cytoplasmic membrane proteins. SOSOI server predictions (Table 6) shows that positively charged amino acids are more at the end of trans membrane region. Protein-protein interaction study has shown some hypothetical proteins are involved in essential cellular process such as transport across membrane, biosynthesis of molecules, translational regulation. Hypothetical protein giÇâ‚ ¬24214908 (Figure 1) interacts with SUA5 protein which is known as one of translational regulator from YrdC/SUA5 family. Search for giÇâ‚ ¬24215909 shown to be involved in chloride transport with chloride channel protein (EriC gene). Protein giÇâ‚ ¬24217373 found to be interacted with S-layer like protein (slpM) which forms layer around bacteria to attach other surfaces and protect it from environment. Additionally it involve in cell devising p rocesses and transport across membrane. Protein giÇâ‚ ¬294827687 had shown interaction with proteins for bleomycin resistance, chorismate synthase (Trp biosynthesis) and Mammalian Cell entry (MCE) like proteins. Figure 2 shows 3D structures of proteins giÇâ‚ ¬24214908, giÇâ‚ ¬24213620, giÇâ‚ ¬24214753, gi|24213945 predicted from amino acid sequence on PS2 server by using templates 1vf8A, 3bo5A, 1f9zA, and c2efsA respectively. Conclusion: Development of potential bioinformatics tools and databases has opened new platform for in-silico study. Currently it is very needful to annotate and characterize hypothetical proteins in Leptospira interrogans serovar. These hypothetical proteins may have an imperative role in producing many virulence factors and cause serious infection or disease. We have analyzed 12 hypothetical proteins from KEGG database and categorized its physicochemical properties and recognized domains and families using various bioinformatics tools and databases. The structures were modeled and their ligand binding sites were identified. Physicochemical predictions made for hypothetical proteins, which can be used to find therapeutic agents against infections caused by Leptospira interrogans. Some of hypothetical proteins serves as channel proteins, ribosomal proteins or are involved in cell cycle process. Families which were identified for these hypothetical proteins are involved in normal cellular process es and the resistance against drugs. Ligand binding hotspots were found with Q-sitefinder which shown amino acids involved in interaction with ligands. It will help in study of molecular docking for development of potent and effective target against Leptospira infection. Acknowledgement: This study was supported by NIPER Guwahati academic staff. We are very grateful for their excellent support in every manner. References: Chou L-F, Chen Y-T, Lu C-W, Ko Y-C, Tang C-Y, Pan M-J, et al. Sequence of Leptospira santarosai serovar Shermani genome and prediction of virulence-associated genes. Gene [Internet]. 2012;511:364–70. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23041083 Langston CE, Heuter KJ. Leptospirosis. A re-emerging zoonotic disease. Vet. Clin. North Am. Small Anim. Pract. [Internet]. 2003;33:791–807. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0195561603000263 Kohn B, Steinicke K, Arndt G, Gruber AD, Guerra B, Jansen A, et al. Pulmonary abnormalities in dogs with leptospirosis. J. Vet. Intern. Med. Am. Coll. Vet. Intern. Med. [Internet]. 2010;24:1277–82. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20738768 Picardeau M, Brenot A, Saint Girons I. First evidence for gene replacement in Leptospira spp. Inactivation of L. biflexa flaB results in non-motile mutants deficient in endoflagella. Mol. Microbiol. [Internet]. 2001;40:189–99. Available from: https://www.scopus.com/inward/record.url?eid=2-s2.0-0035050686partnerID=40md5=ea6dce51e08375c70cdc92eb578e74b1 Adinarayana KPS, Sravani TS, Hareesh C. A database of six eukaryotic hypothetical genes and proteins. Bioinformation. 2011;6:128–30. Hsieh W-J, Pan M-J. Identification Leptospira santarosai serovar shermani specific sequences by suppression subtractive hybridization. FEMS Microbiol. Lett. [Internet]. 2004;235:117–24. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15158270 Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori M. The KEGG resource for deciphering the genome. Nucleic Acids Res. [Internet]. 2004;32:D277–D280. 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Famous graphic designers and illustrators

The most recognized and famous graphic designers and Illustrators are from the US and different countries from Europe, but what about the graphic designers from the Philippines. Kate Morose a famous British artist, and Wham Abaca a Philippine artist who Is not so famous. Two artists that are very good designers and both use pop art and contemporary. Also both work with an explosion of colors. Just looking at their work, you can see the free mind that both have. But Just because Kate Is fromEngland and Wham Is from the Philippines, do not have the same popularity. All around the world you can find people who have an Incredible talent, you do not have to go to England to find good graphic designers. The two illustrators, we just mention, their style is like pop art, with a lot of color and a lot of shapes. Kate Morose use words, with funny letters that make a massage. Wham Abaca, also give us a massage like every artist do, but he just work with images and some time a couple of words. The big difference between this to great artist, are that Skate's work is softer and Wham is most grotesque.Even with this big different, both are good designers that make their ideas flow in their works and looks garish. Skate's and Wham's designs blow up with colors. Kate uses more colors that Wham, she almost use the entire color palette in Just one design and it not looks heavy. But even if Wham is working Just with specific color, all their work is colorful and looks bright. Both make the people turn to see their work, because the bright colors attract their attention. It is difficult to make a good design with a lot of colors, because you have to choose the right tone and make a good contrast when you put them together.Kate and Wham have made a very good Job with that. As you can see in the pictures, both artists try to give massages using their own style and giving life to whatever they want to transmit. Kate gave us very clear what she wants to said, because she uses words, and the words are easy to read. Wham's works Is more like â€Å"one picture tells us more than a thousand words†, because he do not use a lot of words, sometime non. But some of Skate's designs the words are difficult to read and the Wham's pictures are difficult to make a clear message.Even o both reflect their free mind and creatively very well, but not all the people can make a clear message from their creations. As we could saw, designs from a Philippine It Is as well from designs from someone of England. Wham has a unique style, kind pop art, but he put his own touch. He knows how to combine the colors and make them look very attractive, making his wants to give a message, which reflects his thinking. Even if it is look a little bit strange and scary. The world is a place full of persons with great talents that can be found even in places that are not so common like the Philippines.

The Great Gatsby, Invisible Man, And The Adventures Of...

Right Bub Sure it was: The Narrator’s Ability to Control Perceptions From the scrolls in the Library of Alexandria in the third century BC telling the stories of the romans conquering barbarians, to the negative portrayal of political enemies in modern history textbooks, the historian recounting the stories has power over the entire story. Able to describe other seemingly established civilizations in the Americas as savages, European historians were able to control the entire story, portraying themselves as heroes, upon conquering these societies. This narrative power is also present in many examples of modern literature including The Great Gatsby, Invisible Man, and The Adventures of Huckleberry Finn. In these works, the author, like†¦show more content†¦Despite maintaining a powerless position throughout the course of the story, a narrator who imparts his assumptions and perceptions on the descriptions of events ultimately fulfills his need for power by controlling the narrative. Altering the reader’s perception of characters and eve nts, an otherwise powerless narrator is able to fulfill their desire for power. This narrative power is exhibited by the narrators in Junot Diaz’s The Brief Wondrous Life of Oscar Wao. Diaz utilizes different narrators, each with distinct language and perspectives illustrating the possibilities of narrative power. Despite his best efforts to â€Å"share some of his playerly wisdom† (173) and â€Å"fix Oscar’s life†, Yunior is unable to convince Oscar turn his life around and is ultimately powerless with those he cares about(175). Yunior not only failed to convince Oscar to run for more than a few days but also failed on many attempts to persuade Oscar to abort his â€Å"Battle of the Planets routine† (183). This failure to change Oscar’s life is furthered when Oscar â€Å"jumped of the New Brunswick train bridge† (191). Not only had Yunior failed to his â€Å"Oscar Redemption Program† and prevent his attempted suicide but also Yunior was repeatedly rejected by Lola. â€Å"The opposite of the girls [Yunior] usually macked on†, Lola was â€Å"easy to care about† (168). Despite Yunior’s success with many girls

Single Parent Vs Nuclear Family Essay - 847 Words

Single-Parent Families versus Nuclear Families Once in society, it was absurd not to have both parents take care of their children. Parenting, it is a piece of life many people grow up with, and the way it is done keeps changing over time. In today’s society, the two most popular trends in families follow, still, the nuclear family, but now, also the single-parent family dynamic. The single-parent home and nuclear family may seem like they are quite similar, but they do have their differences. Single-parent homes and the nuclear family have differences: financially, affectionately, and just living in general. Financially, single parents have to maintain a steady job in order to support the family. Unlike nuclear families, single parents†¦show more content†¦For example, if one does not have a good job, whether it is low pay or long shifts, and the other parent has the same issue, or is unemployed, they may also feel the same problems as the single parents. 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