Eyanosch Week 11

User Page: Erich Yanoschik Team Page: Oregon Trail Survivors

10 Biological terms
- shigellosis - Shigella infection (shigellosis) is an intestinal disease caused by a family of bacteria known as shigella. The main sign of shigella infection is diarrhea, which often is bloody. (http://www.mayoclinic.org/diseases-conditions/shigella/basics/definition/con-20028418)
- rifampin - a semisynthetic broad-spectrum antibiotic, C 4 3 H 5 8 N 4 O 1 2, used in the treatment of pulmonary tuberculosis, asymptomatic carriers of meningococcal disease, and leprosy. (http://dictionary.reference.com/browse/rifampin)
- rifaximin - is a semisynthetic antibiotic based on rifamycin. It has poor oral bioavailability, meaning that very little of the drug will be absorbed into the blood stream when it is taken orally. Rifaximin is used in the treatment of traveler's diarrhea and hepatic encephalopathy, for which it received orphan drug status from the U.S. Food and Drug Administration in 1998. (https://en.wikipedia.org/wiki/Rifaximin)
- pouchitis - is inflammation of the ileal pouch (an artificial rectum surgically created out of ileal gut tissue in patients who have undergone a colectomy), which is created in the management of patients with ulcerative colitis, indeterminate colitis, FAP, or, rarely, other colitides. Patients with pouchitis typically present with bloody diarrhea, urgency in passing stools, or discomfort while passing stools. The loss of blood and/or dehydration resulting from the frequent stools will frequently result in nausea. Extreme cramping and pain can occur with pouchitis. (https://en.wikipedia.org/wiki/Pouchitis)
- putative - generally believed to be something; commonly accepted or supposed (http://www.merriam-webster.com/dictionary/putative)
- MIC (minimal inhibitory concentration) - the smallest concentration of an antibiotic that regularly inhibits growth of a bacterium in vitro (http://www.merriam-webster.com/medical/minimal%20inhibitory%20concentration)
- type III secretion system - Type three secretion system (often written Type III secretion system and abbreviated TTSS or T3SS, also called Injectisome or Injectosome) is a protein appendage found in several Gram-negative bacteria. In pathogenic bacteria, the needle-like structure is used as a sensory probe to detect the presence of eukaryotic organisms and secrete proteins that help the bacteria infect them. The secreted effector proteins are secreted directly from the bacterial cell into the eukaryotic (host) cell, where they exert a number of effects that help the pathogen to survive and to escape an immune response. (https://en.wikipedia.org/wiki/Type_three_secretion_system)
- transient - lasting a very short time; of a mental act; causing effects outside the mind(http://www.vocabulary.com/dictionary/transient)
- attenuation - reduced especially in thickness, density, or force; tapering gradually usually to a long slender point (http://www.merriam-webster.com/dictionary/attenuate)
- nascent - beginning to exist : recently formed or developed (http://www.merriam-webster.com/dictionary/nascent)
Article Outline
- What is the importance or significance of this work (i.e., your species)?
  - The importance of this work is to identify which genes are induced or repressed depending on the drug tested. These bactrium are responsible for shigellosis, a bacterial infection of the intestine that results in bloody diarrhea and fever. Two antibiotic compounds were studied, rifampin (RP) and rifaximin (RX), both inhibiting RNA synthesis which inhibits growth and reproduction processes of the bacteria. Due to recently increasing resistance to antibiotics in shigella strains it is imperative to see which genes are being inhibited or induced with the current market antibiotics. The expression profiles of micro arrays will provide insight into the interaction between the drugs and bacteria. By inhibiting RNA synthesis, no new proteins will be produced, any proteins still present have yet to degrade.
- What were the methods used in the study?
  - Shigella flexneri 2a strain 301(Sf301) was used in the study. The drugs tested RP and RX were utilized at a concentration of 51.2 mg/mL and diluted with caMHB. The Minimal inhibitory concentrations of the two drugs for Sf301 were determined. Sf301 was taken from a 24 hour culture in caMHB and inserted into caMHB until reaching the desired optical density at OD600. Once the exponential growth phase was reached the drugs RP and RX were added to the cultures to yield final concentrations of 0xMIC, 0.25xMIC, 0.5xMIC, 1xMIC, 2xMIC, and 4xMIC. The drug treatment phase consists of cultures that had drugs added to create 0.5xMIC and 1xMIC with the solvent at 0.25%(v/v). The control cultures had the same final concentration but without the drugs added, solvent at 0.25%(v/v). After 10, 30, and 60 minutes samples were collected and washed for subsequent RNA isolation, each experiment was independently performed 3 times. The RNA was reverse transcribed to prepare a copy of the cDNA that was labeled with flourescent dyes. Cy3-dCTP dye for the control and Cy5-dCTP for the drug treated samples. The genome microarrays were created by purifying the amplified products of ORF-specific primer pairs with the MultiScreen-PCR plates. The products were readjusted to 100ng/microliter and spotted onto gamma amino propylsilan0coated GAPII slides by using Omni-Grid microarrayer. The protocol followed for the hybridization of cDNA to the DNA microarrays can be found http://www.ifr.ac.uk/safety/microarrays/protocols.html#Hybridisations. The slides were scanned by a GenePix 4100A scanner and quantified using GenePix Pro 6.0 software. Local background values were subtracted and the data set was filtered to exclude "not found" features from analysis. MIDAS sofware (http://www.tigr.org/software/tm4) was used to perform normalizations with flourescent signal ratios. The mean Cy5/Cy3 ratios of the gene were calculated to provide further analysis and yield if there were changes in the expression of a the gene. Significant change of expression is if the mean ratio is anything greater than a twofold change eitehr way. qRT-PCR (Quantitative real-time PCR) performed on ABI 7000 instrument using Power SYBR Green Universal Master Mix. Primer Premier 5.0 software designated gene-specific primers. The manufactures recommendations were used for real time PCR. The amount of each gene present was measured relative to a standard transcript, 16S rRNA, and each cDNA was assayed in triplicate PCR reactions.
- Briefly state the result shown in each of the figures and tables.
  - Figure 1. Provides a visual of the growth curve for Sf301 in the presence or absence of two RNA polymerase inhibitors (RX and RP). Figure 2. Provides percentages of genes induced and repressed for each functional class. Figure 3. Shows the relative fold change for the genes in table 1 determined by the quantitative real-time polymerse chain reaction. Table 1. Proivdes a list of Gene-specific primers, both sense and antisense primers, for the quantitative real-time polymerse chain reaction.
- How do the results of this study compare to the results of previous studies (See Discussion).
  - The study aims to provide insight into the inner workings of RNA polymerase inhibitors and bacterial growth. By using two different antibiotics, both of which are RNA polymerase inhibitors in unison and separate to improve the pool of knowledge in regards to bacterial response of antibiotic treatments. Previous studies have shown 1xMIC RP moderately up-regulated expressions of genes related to RNA synthesis, the genes encoding for sigma factor were induced within the study. They also show that target genes were though to be retgulated by growth state and are affected by high drug levels. The number of virulence genes affected was larger than previous studies, the products of many genes repressed may be related to virulence which determines its pathogenicity. The virulence plasmid and the chromosomal encoded SHI-2 island are thought to have been acquired by horizontal transfer.
  1. Describe the experimental design of the microarray data, including treatments, number of replicates (biological and/or technical), dye swaps.