PREP Course # 2 Optimizing Poster Presentations. Presented by: Bettie M. Steinberg, PhD Chief Scientific Officer

Transcription

1 PREP Course # Optimizing Poster Presentations Presented by: Bettie M. Steinberg, PhD Chief Scientific Officer

2 CME Disclosure Statement The North Shore LIJ Health System adheres to the ACCME s new Standards for Commercial Support. Any individuals in a position to control the content of a CME activity, including faculty, planners, and managers, are required to disclose all financial relationships with commercial interests. All identified potential conflicts of interest are thoroughly vetted by the North Shore-LIJ for fair balance and scientific objectivity and to ensure appropriateness of patient care recommendations. Course Director, Kevin Tracey, has disclosed a commercial interest in Setpoint, Inc. as the cofounder, for stock and consulting support. He has resolved his conflicts by identifying a faculty member to conduct content review of this program who has no conflicts. Bettie Steinberg has disclosed support of study drug from Pfizer, Inc. for her clinical trial of Celecoxib. She has resolved the conflict by identifying a faculty member to conduct content review of this presentation who has no conflicts.

3 What Makes a Poster Presentation Different? Visual, not written Esthetics matter: needs to be attractive Present data in simple manner Active audience Drives discussion Will skip altogether if not interested or hard to see Distracting environment Target audience standing Noisy Movement of people, interruptions Variable distance from poster

4 How to Generate Interest in Your Poster Have interesting data Write exciting abstract Have catchy title Make the poster visually exciting

5 Parts of a Poster Title From abstract Poster number sometimes required on same line Authors/Affiliations Nice to use institutional logo Can skip degrees unless required to include Background or Introduction Results (I put methods with results) Conclusions Related presentations this meeting (posters or talks) Not needed: abstract, references

6 The Structure of The Poster Make it easy to read from -5 feet away State information simply Use bullets, not text This is a poster, not a paper Label photographs so easy to understand Number each section and figure in order so readers don t guess order for reading Use lots of color, make it pretty Background can be light or dark, data and text must contrast Red/green don t contrast for color-blind people Don t use high-gloss laminate, reflects glare from lights

7 Recommended Fonts and Sizes Best font is Arial Easy to read, clean Be consistent throughout poster all labels Poster Title: 6 Bold Authors: 5 Bold Affiliation: Bold Headings: 36 Bold Bulleted text: 3 Introduction, figure conclusions, study conclusions :

8 Poster Layout Follow instructions on size and layout Vertical: information goes top to bottom Readers stand in one position Can see whole poster at one time Use one or two columns Don t put most important data on bottom if possible Horizontal: go top to bottom in columns Readers must move to see whole thing Columns should be no wider than ~ shoulder width Number of columns depends on width of poster

9 ppak/pak ppak/pak Relative of P5NFB Relative of IBβ p-pak/ Pak p-pak/ Pak Relative Proliferation Relative apoptosis p-pak/pak p-pak/pak Relative COX- levels Relative I B levels P-p38/p38 levels Example of Vertical Poster Increased Phospho-Pak and Pak Contribute to Rac- Mediated COX- in Recurrent Respiratory s Rong Wu, Allan L. Abramson, Mark J. Shikowitz and Bettie M. Steinberg The Feinstein Institute for Medical Research, North Shore Long Island Jewish Health System.Background Respiratory papillomas premalignant tumors of the airway Caused by HPVs, primarily HPV 6 and HPV The epidermal growth factor receptor (EGFR) overexpressed in papillomas EGFR activates the GTPase Rac, induces Rac overexpression NFB activated in papillomas, due to loss of IB, not IB Cyclooxygenase- (COX-) and its product PGE induced in papillomas COX- elevated in many premalignant and malignant tumors In cancers, prostaglandins immune response, tumor growth, angiogenesis, tumor cell death COX- expression in papillomas requires EGFR and PI3K signaling. Does not require ERK activity Rac protein is required for EGFR-mediated COX- expression, functioning in part through activation of p38 in papillomas, but not in normal cells Question addressed in this study: do the Rac effectors Pak or Pak mediate the activation of p38 and induction of COX- in papilloma cells?. Pak/ Are Constitutively Phosphorylated in Vivo in s N N P P P P 8 5 p-pak/pak 6 Pak 5 Pak Pooled biopsies Phosphorylated Pak/ is nearly undetectable in normal tissue, abundant in papilloma tissue No difference in total Pak/ (not shown) No difference in Pak/ mrna (not shown) and normal laryngeal tissue biopsies were extracted and analyzed by western blot using an antibody that detects both phosphorylated Pak and Pak (Cell Signaling), stripped and re-probed with antibodies specific for Pak and for Pak. RNA was analyzed by Q-PCR, normalized to -actin mrna Results are means SD of the ratio of phospho-pak/ to total Pak in the tissues. s Arrows: location of the basement membrane p-pak/pak Bar: m 3.Pak/Pak are Hyperphosphorylated in Vitro in cells, mediated by Rac. Pak and Pak Contribute to COX- Expression in Cells Through NFB, Not p38 sirna Luc Pak Pak sirna Luc Pak Pak cells cells COX- COX- Pak Pak IkBβ P-p38 p38 actin. Luc sirna Pak sirna Pak sirna Pak Pak IkBβ P-p38 p38 actin 6 Luc sirna Pak sirna 5 Pak sirna 3 Knockdown of either Pak or Pak reduces COX- levels in primary papilloma cells, suggest that both isoforms contribute to induction of COX- Reduction of COX- is only partial, not proportional to loss of Pak/ Knockdown of either Pak affects levels of the other, suggests cross-regulation Knockdown of Pak/ partially restored levels of IB in papilloma cells, which should lead to a reduction in NFB activation but not normal cells Pak and Pak do not mediate p38 activation downstream of Rac in papilloma cells and normal laryngeal cells in KBM were transfected for 6 hr with 5 nm duplex luciferase (Luc) sirna or targeting either Pak or Pak (Dharmacon) by using lipofectamine (Invitrogen), fed with KGM plus sirna for 7 hrs, and cell extracts analyzed for sequential COX-, Pak, Pak, IkB and actin by western blot Blots were quantified by densitometry and normalized to actin. Results are means SD of 3 experiments with normal cells and 3 with papilloma cells, p<.5 compared to controls. 5. COX- Activity Is Required for Maximal Growth and Survival of Cells Luc sirna Pak sirna Pak sirna N P P p-pak/pak PAK PAK actin N P P P5NFB IkBβ actin sirna Luc Rac Luc Rac p-pak/pak Pak Pak Rac Actin cells cells 5 s s lucsirna 7 RacsiRNA s s p-pak/pak are significantly increased in papilloma cells NFBp5 is elevated in papilloma cells compared to normal cells, IB lower sirna knockdown of Rac reduces phosphorylation of Pak/Pak levels in papilloma cells Nearly undectable basal p-pak/ in normal cells Reduction in papillomas significant, reduction in normal cells not significant Results consistent with Pak/ functioning downstream of Rac in papillomas and normal cells cultured in serum free KGM containing EGF and Insulin. Cells extracted and analyzed sequentially for p-pak/, p5 NFB and IB by western blot. and normal laryngeal cells were treated with Rac sirnas for 7 hrs, cells extracted and analyzed by sequential western blot for Rac, phosphorylated Pak/Pak, total Pak & Pak, and actin Results are means SD of experiments with 3 normal and 3 papilloma cultures, p<.5 compared to control Celecoxib (M) Celecoxib (M) Proliferation is reduced in papilloma cell cultures treated with increasing concentrations of COX- inhibitor celecoxib Apoptosis is enhanced in these cultures Minimal effects on normal cells Net effect is significant reduction in papilloma cell number Suggests that inhibiting COX- induction can have therapeutic effects Proliferation: cells cultured in KGM were incubated with increasing concentrations of celecoxib for hrs,.5 g/ml BrdU added for 8 hrs, and cells incorporating BrdU determined by immunohistochemistry using anti-brdu antibody (B-D). Percent labeled cells in fields (approximately 5 cells) was determined and results normalized to control with no celecoxib (approximately %) Apoptosis: cells were cultured in KGM plus celecoxib and apoptosis measured by cytoplasmic presence of nucleosomal fragments using a sandwich ELISA (Cell Death Detection ELISA, Roche) Results are the mean + SD of two separate experiments in duplicate with each type of cell culture p<., p<. 6. Proposed Model for Signaling Pathways That Contribute to COX- Expression in Recurrent Respiratory Cells Other RTKs? EGFR GPCRs? PI3K Rac Rac? Pak/ p38 IB/NFB COX- Cell Survival and Proliferation Supported by grant P5 DC3 from the National Institute on Deafness and Other Communication Disorders, NIH, and a grant from the Frankfort Family Foundation

10 Relative EP mrna Levels Relative Proliferation Relative EP - levels N pool p59 p58 p p97 p39 p653 p698 Relative HPV E7 Levels Percent positive sites Relative mrna levels Cell Death Detection Ave. Wart volume (mm3) Example of Horizontal Poster Prostaglandin E Receptors Regulate HPV Infection in Recurrent Respiratory Cells Rong Wu and Bettie M. Steinberg The Feinstein Institute for Medical Research, North Shore Long Island Jewish Health System. Background Respiratory papillomas premalignant tumors caused by HPVs, primarily HPV 6 and HPV The EGF receptor (EGFR) overexpressed in papillomas Enhanced PI3K activity linked to the EGFR EGFR and PI3K activate the GTPase Rac, induces Rac overexpression Rac induces cyclooxygenase- (COX-) expression COX- and its product PGE contribute to cell proliferation and resistance to apoptosis PGE elevates COX- levels in papillomas through a positive feed-back loop 3. COX- is induced by HPV E7, not E6 Lentivirus Con E6 E7 COX- p - ERK ERK Actin plv plv E6 plv E7 COX- is induced by HPVE7 May act through EGFR signaling, since ERK phosphorylation increased laryngeal cells infected with lentivirus expressing HPV E6 or E7. 8 hours later, cells extracted and analyzed for COX-, p-erk, ERK and actin by western blot 6. (continued) EP mrna was dramatically elevated in papilloma tissues, also increased in normal adjacent tissues of RRP patients, but not normal tissues from control patients Methods and normal laryngeal tissue biopsies were obtained from surgical biopsies RNA was extracted from the tissues, synthesis of cdna was performed, and analyzed by Q-PCR Results are mean SD of four papillomas, three normal adjacent tissues and three normal tissues, relative to GAPDH expression, p<. compared to laryngeal normal, p<. 7. Inhibition of EP induces apopotosis in papilloma cells 7. Celecoxib also reduces wart formation and rate of wart growth UV only UV+celecoxib The activity of PGE is mediated by four G-protein coupled E-type prostanoid receptors (EP -) Question addressed in this study: Which PGE receptor mediates the effects of PGE on HPV transcription and cell viability?. PGE enhances COX- expression, feedback loop enhances HPV expression Control.5 PGE Celecoxib COX- Total.5 protein stain Control PGE Celecoxib HPV E6 HPV E7 Treatment with PGE increased COX- protein levels in papilloma cells Celecoxib (a selective inhibitor of COX- that blocks PGE synthesis) decreased COX- expression PGE enhances HPV expression, celecoxib inhibits it cells treated with PGE at 5 nm or celecoxib at 5 µm, cells extracted after 8 hrs and COX- detected by western blot Results are means SD of primary cells from three different papilloma patients and three normal tissues Total proteins visualized by fast green stain as loading control 5. E-type prostanoid receptors (EP - ) are expressed in papilloma tissues (M) 5 5 EP antagonist Inhibition of EP, but not EP, EP or EP 3, induced apoptosis in papilloma cells in a dose-dependent manner EP agonist further suppressed the normal low apoptotic level in papilloma cells, which could reflect a response to the endogenous PGE made by the cells cells in serum-free medium were treated with EP - agonist (3 nm), antagonist (M), or antagonist (M) for 5 minutes then adding PGE (5 nm) or vehicle for hours, apoptosis measured by nucleosome release into the cytoplasm Results are means SD of 3 experiments with papilloma cells from 3 different patients, p<. compared to controls. 8. EP affects HPV transcription weeks Celecoxib reduced induction of E6/E7 mrna by 95% Celecoxib decreased wart number and size Rabbits with latent CRPV infections split two treatment groups:. saline (as a negative control). oral celecoxib (5 mg/kg) for duration of experiment Irradiate each site with. J/cm- UV-B Monitored weekly for appearance of warts, and wart length, width and height measured Results analyzed by Chi-square, p<.5 8. Exogenous PGE overcomes celecoxib inhibition, activates latent infection and promotes wart growth E6/E7 Induction Control 5 P<.5 PGE 3. PGE stimulates proliferation and celecoxib inhibits apoptosis in EP papillomas papilloma cells, but not in normal cells EP PGE (nm) Relative Apoptosis EP 3 6 normal EP papilloma 5 Fast green stain EP EP EP 3 EP 3 All four EP subtypes are expressed in papillomas EP, EP, and EP below detectable levels in normal tissue, EP 3 was expressed EP 3 was not elevated in papillomas compared to normal tissue EP highly elevated in papillomas Tissues were extracted from pool of 6 normal tissues and from 7 papilloma tissues and analyzed for EP by western blot Celecoxib (µm) Results are means SD, relative to total proteins visualized by fast green stain PGE stimulated papilloma cells growth, not normal cells Celecoxib induced a dose-dependent increase in apoptosis in papilloma cells, not normal cells 6. EP mrna levels markedly increased in both papilloma tissue N. Adj and normal laryngeal tissue biopsies were obtained from surgical biopsies, used to establish primary cell s cultures and normal cells cultured in serum free KGM containing EGF and insulin cells treated with PGE at, 5, 5 and 75 nm for 8 hrs, cell growth measured by MTT assay and normal cells treated with celecoxib at.5, 5., or 7.5 M or vehicle for hours, apoptosis measured by nucleosome release into the cytoplasm Results are means SD of experiments with 3 normal and 3 papilloma cultures, p<. compared to control EP 3 EP EP EP con EP antagonist EP antagonist EP 3 antagonist EP antagonist Inhibition of EP reduced HPV E7 transcription, but not EP -3 cells in serum-free medium were treated with EP - antagonist (M) for 3 min, RNA extracted from the cells and analyzed by Q-PCR Results are means SD of different patients with papilloma cells p<. compared to controls 9. Conclusions The EP receptor for PGE plays a major role in HPV-infected papillomas Inhibition of EP could potentially be an effective therapeutic target EP HPV EGFR COX- PGE growth and survival Celecoxib DMSO PGE Wart formation Number warts (number of inoculated sites) treatment weeks percentage DMSO () 5 PGE 5 (5) PGE restored mrna activation to 75% of level seen previously in rabbits without celecoxib PGE also restored formation of warts Celecoxib acting through inhibition of COX-, not off-target effect Oral celecoxib treatment (5 mg/kg) for weeks UV-irradiate latent CRPV sites Treat subset of sites daily for 5 days with µl PGE ( µm dissolved in DMSO). Control sites treated with DSMO After weeks, count appearance of small warts, analyze all sites for viral RNA by RT-PCR Results analyzed by Chi-square test, p<.5 8. Conclusions COX-/PGE is necessary for activation of latent papillomavirus infection Celecoxib is an effective inhibitor of activation PGE may be a potent activator, since wart appearance with UV plus topical PGE treatment appeared to be faster than with UV alone

11 The Title Make it the main take-home message Example: Rac and Cox- are Constitutively Expressed in the Airway of Recurrent Respiratory tosis Patients vs. Analysis of Rac and Cox- Expression in Recurrent Respiratory tosis

12 Introduction Background information, sets up study Bullet format. Can add figure or cartoons Not the abstract. Background Respiratory papillomas premalignant tumors of the airway Caused by HPVs, primarily HPV 6 and HPV EGFR activates the GTPase Rac, induces Rac overexpression NFB activated in papillomas, due to loss of IB, not IB Rac protein required for EGFR-mediated COX- expression Functions in part through activation of p38 in papillomas, not normal cells Question addressed in this study: do the Rac effectors Pak or Pak mediate the induction of COX- in papilloma cells

13 Presenting the Data P-PAK/PAK P-Pak/Pak. Pak/ Is Constitutively Phosphorylated in Vivo in s P-Pak/ Pak Pak N N P P P P Phosphorylated Pak/ very abundant in papilloma tissue Nearly undetectable in normal tissue More Pak in normal tissue than in papilloma tissue and normal laryngeal tissue biopsies analyzed by western blot, using antibody that detects both p-pak and Pak (Cell Signaling), stripped and re-probed with antibodies specific for Pak and Pak Graphs: Relative mean SD of the ratio of phospho-pak/ to total Pak or Pak, p<.5 6

14 Presenting Images Don t use footnotes Label figures completely Rac Stain Tissue Clinically Tissue Tracheal papilloma

15 Conclusions Can be bullets, diagrams or combination 6. Proposed Model for Signaling Pathways That Contribute to COX- Expression in Recurrent Respiratory Cells PI3K Rac? EGFR Cells Rac Pak/ p38 IB/NFB COX- Cell Survival and Proliferation

16 At The Poster Session Multiple purposes Present your study Get feedback from experts Identify potential collaborations Know who you are talking to Their background and interest What lab they are in (your competition?) Discuss their poster/talk

17 At The Poster Session part Be prepared to give out your contact information Multiple approaches work Handout card with your name/ /phone # Printout of your poster with above info on it Get contact information from people interested/note their research areas Have a small notebook and bag available Wear comfortable shoes Have a great time!