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50 Cards in this Set
- Front
- Back
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What are antibacterial peptides? |
antibacterial or cationic peptides, discovered 20 years ago in skin of frogs -have been over 700 discovered -innate immune mechanism, with potent broad spectrum antimicrobial agens -active against fungi, bacteria, viruses and parasites |
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antimicrobial peptides structure |
12-50 aa length, cyclical -net positive charge and have ~50% hydrophobic residues -show activity against antibiotic resistant bacteri
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What are the antimicrobial peptides mechanism of action? |
-interact with charge on LPS and displace Mg, they then insert into membrane and form a channel which disrupts the membrane and binds internal targets |
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What are the 3 ways antimicrobial peptides cause bacterial cell death? |
1. disrupt outer membrane integrity 2.disrupt inner cytoplasmic membrane integrity 3. bind DNA |
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What does the bacteria need to have to have immunity to antibacterial peptides? |
bacteria that produce antimicrobial peptides against another bacteria are immune themselves. gene cluster with the antibacterial peptide also contains membrane associated immunity protein(+,hydrophobic and protects bacteria from peptide produced), ATPase and an ABC transporter |
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What kind of bacteria produce an antimicrobial peptide against Clostridium and Listeria? |
Firmicutes produce bacteriocin |
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Why was the development of resistance against antimicrobial peptides thought to be improbable? |
-it would require alteration of bacterial membrane -energeticaly too costly -but it does occur! |
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How does salmonella confer resistance to antimicrobial peptides? |
-invade macrophages and the presence of an antimicrobial peptide activates a 2 component system (PhoP/PhoQ) |
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How does the PhoP/PhoQ system work when there are no microbial peptides? (repressed state) |
Mg2+ binds PhoQ pocket to repress its activity so that no PhoP is activated |
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How does the PhoP/PhoQ system work in the active state, when there are antimicrobial peptides present? |
antimicrobial peptides displace Mg2+ and bind the pocket of PhoQ changing its conformation. It dimerizes and autophosphorylates, and transfers a P onto PhoP which activates PagP gene transcription |
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What is the pagP gene? |
-controlled by PhoP/PhoQ -phospholipase/palmitoyltransferase which removes a palmitate from the OM phospholipid and transfers it to lipid A(increasing # of chains) -increase interaction btw two leaflets of membrane=more resistant -antimicrobials cannot insert into membrane as easily |
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How does yersinia confer resistance to antimicrobial peptides? |
2 diff forms: -at RT the O Ag chain is long and makes OM less permeable to antimicrobials(O Ag chain upreg = RosA/B pump downreg) -at 37(in humans) O Ag chain is short which is more permeable, so RosA/B pump is upreg to move antimicrobials out of the cell protecting DNA |
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How does the RosA/B efflux pump work? |
-RosA is the shuttle and RosB moves potassium out of the cell and lets H+ ions in -more H+ ions in decreses cytoplasmic pH stopping the antimicrobials/pumping them out of the cell through RosA -prevents interaction of peptides with DNA |
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What are some techniques for studying bacterial pathogenesis? |
using the genome, transcriptome, proteome, and metabolome |
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What is Next generation sequencing? |
-first reported in 2005 -inexpensive and easy way to sequence bacterial genomes -30GB of sequence/week for 15000 (human genome 3GB took 5 years and cost 3,000,000,000) |
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What is comparative genomics used for? |
comparing genome of virulent and avirulent bacterial strains -looking for missing or altered strains |
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What is an open reading frame? |
region of an organisms genome that contains a long stretch of DNA that could potentially encode a protein -need an RBS,start codon,stop codon and no stop codons within the ORF |
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How do you use microarray technology? |
-oligonucleotides attached to glass slide, representing each gene, four spots made for quality control -to compare 2 strains of DNA treat with DNAse which causes nicks and add DNA pol1 which has exonuclease activity so you get excision of some nucleotides on second strain of DNA, fluorescently label DNA strands w/ diff colour, hybridize to microarray |
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How can you use a microarray for an expression analyses? |
-grow up bacteria under one condition, and another with another condition ie. in vivo/in vitro -convert mRNA to cDNA and fluorescently label them, hybridize to probes on microarray slide and read results |
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What kind of output do you get from a microarray reader? |
-2 diff colour labels usually green and red - yellow spots showing up means that both probes have hybridized to one spot -this give insight into proteins produced under certain conditions |
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What is IVET? |
in vivo expression technology -identifies bacterial gene that are expressed in vivo but not in vitro -uses reporter system with promotor trap
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What is the process of IVET? |
1.clone DNA from S.typhimurium into IVET plasmid, inserts before purA LacZY operon and electroporate into E.coli 2. mate with S.typhimurium purA-recipient(suicide plasmid can only survive if integrated into c'some) select for Amp resist colony 3.look at amp plates: Lac+ inserted region of DNA expressed in vivo, infect mice with bacteria
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What are you looking for after you infect mice with bacteria in IVET? |
-isolate bacteria from organs, plate, Lac- colonies (which were lac+ in mice) will be induced in vivo -do blue/white screening to find Lac- colonies
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What are you going to do with the colonies that were Lac- after isolation from mice in IVET? |
1. sequence purA junctions 2.BLAST studies in GenBank 3.Computer analysis |
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What is proteomics? |
study of the proteome using technologies of large-scale protein separation and identification |
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What is the proteome? |
entire complement of proteins expressed by a cell,tissue or organism at a given point under defined conditions
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Proteomics vs Genomics |
-no complete proteome sequenced: genome is stable but proteome is complex
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What variable contribute to the complexity of proteomics? |
-abundance of protein -modifications or truncations of proteins -presence or absence of proteins -different protein profiles based upon altered culture conditions or growth in vivo |
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Why is proteomics so important? |
because of things like tissue tropism of certain organisms: -chlamydia: tissue specific strains exhibit diff tropism -eye or urogenital tract -99.6% nucleotide identity btw strains |
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what are two different important factors to analyze at proteome level? |
1. distinguish btw all possible interaction in all proteomes and actual interactions that occur in one proteome 2. protein quantity in a proteome |
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What are 3 techniques used in proteomics? |
1. 2D gel electrophoresis 2. Mass-spectrometry 3.ITRAQ |
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What is 2D gel electrophoresis? |
-separates protein based on isoelectric point and then by molecular weight -isoelectric focusing: net charge of protein depends on pH of environment, protein stop migrating at pI SDS-PAGE: migration depending on mol. weight |
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How do proteins migrate in the isoelectric focusing gel? |
-carrier ampholytes establish gradient in gel (those with highest pI will migrate towards the cathode) -positively charged protein move toward cathode (-) -negatively charged proteins move toward anode (+) |
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How doe proteins migrate in the SDS-PAGE gel ( 2nd dimension) |
-SDS reduces protein to primary structure independent of charge PAGE: allows proteins to separate based on molecular weight (larger at top) |
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What is Mass spectrometry? |
2 key components: proteins can be broken into peptide fragments by proteases, all peptides are sorted based on mass(m) to charge(z) ratio |
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How do proteases break protein into peptide fragment? |
-trypsin is a serine protease that cleaves after lys and Arg residues |
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What is the difference btw MS and MS/MS |
MS only does ionization and separation, but tandem MS ( MS/MS) also does activation and mass determination |
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How does MS work? |
-ionization: peptides ionized by MALDi or electrospray Separation: via TOF which determines m/z ratio and identical peptides sent to activation chamber (stop after MS if sequence known)
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How does MS/MS work? |
Activation: 2 identical peptides from separation sent into argon-filled chamber and the peptide broken into 2 pieces with a charge (amino=b and carboxyl =y) Mass determination: determines the m/z ratio for mixture of ionized peptide fragments-do a database search to find protein |
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What are some limitations of 2D gel electrophoresis? |
-difficult to resolve: they are basic proteins, small peptides and large proteins, difficult to solubilize -proteins smear and overlap -difficult to get absolute quotation for certain proteins |
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What is ITRAQ? |
isobaric tags for relative and absolute quantitation |
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What do the isobaric tags have? |
a reporter group and a balance group -tag mass is consistent and so reporter group has a strong signal in MS/MS to determine abundance of peptide |
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How does ITRAQ work? |
1.prep different samples and then digest with an enzyme(trypsin) and add diff tags 2. combine samples 3. cation exchange fractionation 4. reverse phage separation 5. LC/MS/MS |
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What is ITRAQ used for? |
-perfect for analyzing bacterial pathogenesis at the proteome level -ie. can look at chlamydia's tissue tropism, genome almost identical so can look for protein expression amount to see differences |
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ITRAQ duplex experiment? |
using different reporter tags ( and a balance so that the mass of each tag is equal) you can do MS/MS on both peptides from each chlamydia strain -shows that the ocular specific strain has one protein present at 3 fold higher (fibrillarin) |
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What is systems biology? |
"bottom up approach" -DNA and protein data analysis for characterization of high level processes and make the hypothesis later after all data analyzed |
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3 principles for systems biology? |
1. list of indiv changes from exp data 2. develop network diagram to see how changes relate to system (determine functional relationship) 3. math or computational model to test correctness of system |
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What is different in systems biology compared to traditional approaches? |
-results are high throughput -requires intensive computational analysis -not hypothesis driven(collect data without hypothesis) -post-exp data analysis results in hypothesis creation and testing |
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Systems biology approach/map |
genomics,transcriptomics,proteomics,metablomics-->data collection-->data mining analysis-->computational network --> devise hypothesis-->data minding-->etc. |
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key difference btw systems biology and conventional biology or cell biology analyses? |
study all genes and proteins at one time and determine hypothesis, get a better idea of factors that contribute to disease |
