Project SEED Poster and Report Tips & Guidelines indyprojectseed/2014/poster.htm

1. Project SEED Poster and Report Tips & Guidelineshttp://www.indyprojectseed.com/2014/poster.htm

2. DEADLINES!!!! • Abstract: Thursday, July 3rd, at 5 PM • E-mail to Amitha (indyseed.intern@gmail.com) to edit by June 30th(optional) • Poster: Thursday, July 17th, at 5 PM • Ideal Target Tuesday, July 15th at 5 PM • E-mail to Amitha (indyseed.intern@gmail.com) to edit by July 14th(optional) • Must be done by deadline or your poster will not be printed!!! • Report: Tuesday, July 22th,at 5 PM • E-mail to Amitha (indyseed.intern@gmail.com) to edit by July 19th(optional)

3. Abstract Guidelines • See WORD Document and template to fill in: • Purpose: A brief summary of your project • Length: 275 words max. • Font: Calibri • Title: size 16, bold • Authors : your name first and bolded, size 14 • Institution: superscripts link authors, size 12 • Body: size 12, justified, single-spaced • See EXAMPLES on following slides

4. Abstract Example 1 The role of NADPH oxidase in age-related collateral growth impairment Nandita Chittajallu1, Mary J. Wenning2, Bruce Henry2, Matthew DiStasi2, Randall G. Bills2, Joseph L. Unthank2, Steven J. Miller, PhD 1Pike High School, Indianapolis, IN, and 2Department of Surgery, Indiana University School of Medicine, Indianapolis, IN Due to cardiovascular disease or injury, arteries may become blocked, decreasing blood flow and thus oxygen delivery to tissues and organs. However, pre-existing arteries that bypass the blockage site may enlarge to compensate for the decreased blood flow. This process, known as collateral growth, is dependent on the production of nitric oxide which is stimulated by decreased blood flow in the bypass arteries. Collateral growth is impaired during aging, and this occurs in part because of an increase in reactive oxygen species (ROS) which interfere with nitric oxide production. The major source of ROS produced in arteries is thought to be an enzyme called NADPH oxidase, which exists in several forms and locations in the artery wall. The goal of this project was to determine how the major types of NADPH oxidase regulate collateral growth by measuring temporal changes in their expression/activity and localization during flow-mediated vascular remodeling. By using quantitative PCR, it was determined that the basal arterial expression of Nox4 and p47phox increases with age. There was also a significant increase p47phox in aged rat collaterals at 7 days in comparison to those of young rat collaterals. This suggested that they are the sources of chronic oxidative stress and therefore inhibitors to collateral growth. This knowledge will allow for the development of targeted approaches to promote collateral growth in aged patients. (Word count: 223)

5. Abstract Example 2 Expression of N-cadherin and β-catenin in Fanc deficient MSCs Terry Ming1, Yongzheng He2,3, Feng-Chun Yang, M.D., PhD2,3 1Carmel High School, Carmel, IN, and 2Department of Pediatrics, 3Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN Fanconi anemia (FA) is a recessive genetic disease caused by mutations of different FANC (Fanc) genes. FA has manifestations such as eventual bone marrow failure and susceptibility to a multitude of cancers. Although previous studies have shown that the loss of FANCG (Fancg) gene in mice decreases the ability of mesenchymal stem cells (MSCs) to support adhesion of hematopoietic stem cells (HSCs), little is known about the molecular underpinning of the pathologic changes. Given the fact that N-cadherin and β-catenin have been shown to be critical for cell-cell interaction and intracellular signaling pathways, in the current study, we investigated N-cadherin and β-catenin expression at both message RNA and protein levels. Our results show that both mRNA and protein levels of N-cadherin expression is lower in Fanc deficient mice. These results suggest that the lower expression of N-cadherin protein may be associated with less HSC-MSC interaction which leads to anemia. (Word count=149)

6. Poster Guidelines • Poster template: http://www.indianactsi.org/template • Include logos: http://www.indyprojectseed.com/2012/logo.htm • Size: Standard: 36h x 48w; Large: 42” h x 48”w. Ask if need larger! • Font: Calibri or other Sans Serif (This notThis); ask if you can’t tell • Font size Recommendations. May need to adjust at end to make fit • Title: 76-96 pt. • Authors: 48-60pt. • Institutions: Same size as Authors or 6pt smaller font if space needed • Headings: 38-50 pt or about 10pt smaller than author • Text: 28-40 pt. or about 10pt smaller than headings • Captions: 22 pt - 34pt or about 6pt smaller font than text. • Colors: Avoid yellow text, font colors always darker than background colors; use a WHITE background for poster but may color fill text inserts. • If use colors, be consistent so it looks professional. • Respect white space!!!!

7. Poster Content • Title • Authors • You first • Others in rank of contributions • P.I. last • Institutions • Overview • Use the same wording as your abstract Formatting same as abstract

8. Poster Content (Cont.) • Introduction • Define all terms • Include only relevant background info • Objective • Purpose of project? • Why would the audience care about your project? • Methods • Simple, exact • Equipment/analysis tools

9. Poster Content (Cont.) • Results • Figures and pictures work best • Only data, not analysis • Discussion/Conclusion • Analyze data; tell us the significance of your results • Relate data back to project objectives • Limitations • Future work • Optional • Literature review • References to other work if at all applicable. • Anything else your lab wants you to add

10. Poster Content (Cont.) • Acknowledgements( last box at the bottom) • Include: “I would like to thank the Indianapolis Project SEED Committee and Staff, the Indiana Section and National membership of the American Chemical Society, and the Indiana CTSI. This research was made possible in part by Grant Number UL1 TR001108 (A. Shekhar, PI) from the National Institutes of Health.” • List other grants / mentors your lab may want to credit … in addition to your department and school – example: Department of Biology, IUPUI School of Science Grants received (include those that funded your project even if you didn’t apply for them yourself. Ask your mentor if you need to recognize any of their grants / department / chairs) All lab members your Mentor approves you to recognize. *ONLY include the Committee and/or Staff Names inside the [brackets] if you have extra space. IF space allows, include ALL committee names and if more space, include ALL staff names

11. Title Authors Institutions Overview Methods Results (cont.) Introduction & Objective Discussion/ Conclusion Results Acknowledgements

12. Title Authors Institutions Overview Introduction & Objective Methods Results Discussion/ conclusion Results (cont.) Acknowledgements

13. Examples • See Website templates and Gigi’s 2011 poster • Nandita’s poster 2010 http://www.indyprojectseed.com/2011/NanditaChittajalu2010.pdf

15. Characterization of a small RNA and its effects on Escherichia coli resistance to a DNA-damaging chemical Jennifer Yu, Kyle M. Hetrick, Kimberly A. M. Storvik, and Patricia L. Foster Department of Biology, Indiana University Bloomington GOAL: This ongoing research investigates the mechanism by which polyphosphate kinase (Ppk) and the small RNA RydD affect NQO resistance (NQOR) in E. coli as well as the phenomenon of NQOR in general. The goal of this project is to screen a mutant library in order to identify genes that affect NQOR. The genes of interest are those in which suppression by RydD is lost. Identification of these genes will provide a better understanding about the contributions of RydD and Ppk to NQOR and DNA Polymerase IV (Pol IV) activity. RESULTS: • INTRODUCTION: • Like all organisms, Escherichia coli (E. coli) must pass on its genetic information in order to survive. DNA is frequently at risk of being damaged by hazardous agents such as ultraviolet radiation and various chemicals. These hazards can create lesionsin the DNA, which • prohibit DNA replication by the primary DNA polymerase • can be fatal if DNA replication is not restarted • Y-family DNA polymerasesare able to replicate past the nucleotide adducts in a process calledtrans-lesion synthesis (TLS) (Fig. 2). In response to DNA damage, E. coliupregulates about 40 genes, including a gene that encodesDNA polymerase IV (Pol IV) in E. coli. Pol IV • is a Y-family polymerase • is not processive, meaning it can only synthesize short stretches of DNA • is error-prone, meaning it can incorporate incorrect nucleotides • has the highest expression among the five E. coli DNA polymerases during DNA damage • Because Pol IV is error-prone yet abundant, Pol IV activity must be highly regulated to prevent widespread mutagenesis in the genome. • one regulator of Pol IV is ppk, which encodes polyphosphate kinase (Ppk) • Ppk synthesizes long chains of phosphates called polyphosphates (polyP) • therefore, polyP levels may affect Pol IV activity • In this project, the TLS activity of Pol IV was assayed by testing the resistance of E. coli to a DNA-damaging chemical, 4-nitroquinoline-1-oxide(NQO), which interacts with DNA bases to form bulky lesions. • ppkmutants exhibit decreased resistance to NQO (NQOR) • when a region called Spk(standing for “suppressor of polyphosphate kinase”) is overexpressed, NQOR is restored • this phenomenon is called suppression • Within the Spkregion, RydD, a small • RNA (sRNA), is encoded. Small RNAs • are short, untranslated RNA molecules • regulate stress responses by binding to messenger RNA molecules, thereby changing the expression of one or more target genes • In Fig. 4, candidates that showed a phenotype similar to WT and ppk-/pSpk++ • had normal NQOR • did not have an interrupted gene that contributed to NQOR • Candidates of interest were those that showed a phenotype similar to the ppk- strain • had decreased NQOR • had an interrupted gene that contributed to NQOR Fig. 4 Fig. 1 E. coli (shown above) is a nonpathogenic model organism. • METHODS: • Prepared a λNK1324 lysate • the lysate contained a λNK1324 bacteriophage, a type of virus that can infect E. coli • the DNA of λNK1324 contains a transposon called mini-Tn10::Cm • a transposon is a section of DNA that can insert itself in random locations within an organism’s genome and inactivate individual genes • this characteristic was taken advantage of in making the insertion mutant library, which is a collection of bacteria, each with one transposon-inactivated gene • Created an insertion mutant library • the transposon was introduced into ppk mutants overexpressingSpk using the λNK1324 lysate • this process, called transposition, allowed the transposon to insert itself in random locations in the E. coli genome • Replica plated • plates of transposed cells were replica plated onto LB + carbenicillin (carb) and LB + 0.1 mM NQO and carb agar plates • antibiotics were used to select for the presence of the Spk plasmid and the transposon insertion • Maintained candidates • candidates of interest were those that demonstrated a decreased NQOR phenotype, meaning there was decreased survival of colonies upon exposure to NQO • they were transferred onto LB + carb and chloramphenicol (cam) agar plates and maintained at 4°C • High throughput NQO assay • a short range of dilutions (10-3 to 10-6 ) was spotted onto LB + 12 μM NQO and carb agar plates, whichenabled high quantity testing of candidates • Traditional NQO assay • a full range of dilutions (10-1 to 10-6) was spotted onto LB + 12 μM NQO and carb agar plates, which enabled higher quality and higher accuracy testing of candidates that showed decreased NQORin the high throughput assay • Located site of Tn10 insertion • arbitrary-primed PCR (AP-PCR) was used to determine the locations of the transposon insertions and thus identify the interrupted genes • the AP-PCR used random-sequence and specific-sequence primers to amplify DNA around the transposon insertion sites • the AP-PCR products were sequenced, and the resulting sequences were compared to a database of DNA sequences using a program called BLAST in order to identify the interrupted genes • the protein products of these genes and their functions were determined using a database of E. coli genes called EcoCyc Key Fig. 5 • Genes revealed by the screen so far are shown in Fig. 5. • Some are membrane proteins, which may play a role in the transportation of NQO molecules across the cell membrane • Some have no known function, only predicted functions Fig. 2 (A) The primary polymerase encounters a lesion and stalls. (B) It falls off the DNA, and Pol IV is recruited to the lesion. (C) Pol IV synthesizes DNA past the lesion. (D) Pol IV is not processive and falls off the DNA shortly. The primary polymerase returns to the DNA and continues DNA replication. • CONCLUSION/FUTURE WORK: • It is still not known whether or not the identified genes are targets of the small RNA RydD and whether they function in a NQOR pathway that includes ppk. • Future work will include mutating the identified genes in various genetic backgrounds (including wild-type, ppk- and ppk-/pSpk++) and testing the NQOR of the resulting strains. • The results of these assays will reveal whether the decreased NQOR these mutants exhibit depend upon ppk or Spk. • Thus, these assays will help us understand how the identified genes contribute to NQOresistanceand DNA Polymerase IV TLS activity, and they will help to determine whether these contributions are related to Ppk or RydD. Fig. 3 • REFERENCES: • Das, S., et al. 2005. “An improved arbitrary primed PCR method for rapid characterization of transposon insertion sites.”Journal of Microbiological Methods63: 89-94. • Jarosz, D.F., et al. 2006. “A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates.”Nature439: 225-228. • Kleckner, N., et al. 1991. “Uses of transposons with emphasis on Tn10.”Methods in Enzymology204: 139-180 • Stumpf, J.D., and Foster, P.L. 2005. “Polyphosphate kinase regulates error-prone replication by DNA polymerase IV in Escherichia coli.”Molecular microbiology57(3): 751-61. • Zhang, A., et al. 2003. “Global analysis of small RNA and mRNA targets of Hfq.”Molecular Microbiology50(4): 1111-1124. The strain used in this project was a ppk mutant that had Spk (containing RydD) overexpressed on a plasmid. ACKNOWLEDGEMENTS: KeunJu Kim, Thomas Sladewski, Jeffrey Stumpf, Hutton Honors College Research Grant to J. Yu, Cox Research Scholarship to J. Yu, and USPHS Grant NIH-NIGMS GM065175 to P.L. Foster

16. Report Content • Abstract • Short summary of your project • Same headings as your poster • There’s no extra work; if you can write your report, the poster will come easily • Afterward (optional) • Reflection on your experience in the program • How will SEED help you with your future (ie: college, career choice/development, etc.)?

17. References!!!!! • Footnotes • You may use footnotes or endnotes to reference.1 • At the bottom of the page: 1Journal of the American Medical Association • Reference section • Reference section1 • Number in order of reference appearance • Separate page: References • 1. Journal of the American Medical Association

18. Signature Section • Must be signed by both parties by Mentor signature by Tuesday, July 22nd, at 5 PM This document prepared by: John D. Smith, Project SEED student, Summer 2014. __________________________Date: __________ (signature) Reviewed by: Dr. Jane B. Doe Project SEED preceptor, Summer 2014 ______________________ Date: ___________ (signature)

19. More Info & Examples • http://www.indyprojectseed.com/2014/poster.htm • Ask Mr. Sanders, Mrs. Harrison, Ms.Montgomery, Brent, or Amitha