PRIMARY RESEARCH ARTICLES

General Instructions:

E-mail your rough draft (due Wednesday, week 3) as an attachment to me. Use Word to write your paper and attach your paper as a Word document. Word is available on all campus computers. Do not use another word processing program; Word is easily accessed by all students on campus. Do not include any personal information on the attachment. I will know who the paper belongs to by the e-mail address. I will randomly send you another student's draft for you to review. The student's name will not be on the paper you are reviewing.. You need to personally review and critique this paper; you may take your own paper for assistance to the Writing Resource Center in the first floor of the library but not the paper you are assigned to review. E-mail your review back to me by the following Friday, 9 AM. You will receive the student review of your own paper plus my review of your paper. You have the weekend to revise your own paper using the suggestions you receive from both your reviewers.

You will not receive credit for your own paper unless I receive your review and it addresses the following questions:

1. What is the writer trying to accomplish with the paper. Is the purpose of the paper clear? Does the paper get right to the point?

2. What questions or concerns do you have about the paper? Are there sections that are difficult to follow? Is the organization, content, flow, and level appropriate for students in BIO 103?

3. Does the paper use correct punctuation? Are there any redundant words, empty or overly complex phrases? Are there confusing or ambiguous phrases? (Avoid the use of this, that, which, and it!) Are there incomplete sentences, run-on sentences?

4. What suggestions do you have for the writer?

5. What did you like about the paper? What are the strengths of the paper?

Keep these questions in mind when you write your own paper and read the scoring rubric for these papers carefully! Also, check out the website on Plagiarism at the University of Indiana. Jen Rouse, Director of the Writing Studio, will be coming to class to help you with this paper. She is a valuable resource; take advantage of her expertise.

Writing Papers in Science

Scientists communicate their work through formal written papers in scientific journals and through oral presentations and posters presented at scientific meetings. A paper describing original work, such as the cytotoxicity experiment you are doing, is called a primary research article. The articles you will read in this course are considered secondary references or papers because they summarize the results of a number of primary papers on a particular topic. You will write a primary research paper discussing your cytotoxicity experiment. There are 6 basic questions addressed in a primary research paper:

1. Why did you study this problem?
2. What did you do?
3. How did you do it?
4. What did you find?
5. What does it mean?
6. How does it relate to previous work in the field?

Most primary research papers follow a set format in answering these questions: a title, an abstract, and four sections including: introduction, materials and methods, results and discussion.To help you understand how to write this paper, the following is an example of the format for an article that appeared in the Proceedings of the National Academy of Science (Proc. Natl. Acad. Sci. USA, Vol. 98, Issue 9, 5317-5322, April 24, 2001). The bracketed comments in red are explanations for your information. Don't worry if you don't understand the scientific content of the paper; it serves as a model only and is an example of an actual study dealing with cell cultures and cytotoxic agents that includes many things you will need in your paper. Of course, your paper will not be this technical and may or may not include detailed figures such as those in this article. Your paper should be at least 3 to 5 pages long. A one page paper is obviously not acceptable. Also, your paper should be double spaced, font 12, Times New Roman, with 1 inch side margins and 0.75 inch top and bottom margins. Figures/Tables and Literature Cited (references) are not included in the 3 to 5 page length. Do not imbed your figures/tables in the text of your paper; include them as separate pages at the end of the document.

[Title: a short description of the experiment and paper] Direct toxicity of nonsteroidal antiinflammatory drugs for renal medullary cells
[Keywords used by search engines] (acetaminophen / aspirin / salicylic acid / indomethacin / caffeine)

[Authors: individuals that helped to design, perform and/or analyze the experiment] Gerson M. Rocha*,, Luis F. Michea*,, Eugenia M. Peters*, Martha Kirby, Yuhui Xu§, Douglas R. Ferguson*,¶, and Maurice B. Burg*,
[Addresses for authors] * Laboratory of Kidney and Electrolytes Metabolism, Hematology Branch, and § Microscope Core Facility, National Heart, Lung, and Blood Institute, Bethesda, MD 20892; and ¶ Department of Pharmacology, University of Cambridge, Cambridge CB2 1QJ, United Kingdom

[Abstract: a short summary of the entire paper. The abstract is a condensed version of each of the four sections of the main paper. The abstract should give the reader a clear idea of the reason behind the experiment, how the experiment was performed and what was found.] Antipyretic analgesics, taken in large doses over a prolonged period, cause a specific form of kidney disease, characterized by papillary necrosis and interstitial scarring. Epidemiological evidence incriminated mixtures of drugs including aspirin (ASA), phenacetin, and caffeine. The mechanism of toxicity is unclear. [These first sentences state the reason(s) for performing the experiment.] We tested the effects of ASA, acetaminophen (APAF, the active metabolite of phenacetin), caffeine, and other related drugs individually and in combination on mouse inner medullary collecting duct cells (mIMCD3). [The previous sentence explains concisely what was done in the study. The next sentence is a summary of the results of the study.] The number of rapidly proliferating cells was reduced by 50% by 0.5 mM ASA, salicylic acid, or APAF. The drugs had less effect on confluent cells, which proliferate slowly. Thus, the slow in vivo turnover of IMCD cells could explain why clinical toxicity requires very high doses of these drugs over a very long period. Caffeine greatly potentiated the effect of acetaminophen, pointing to a potential danger of the mixture. Cyclooxygenase (COX) inhibitors, indomethacin and NS-398, did not reduce cell number except at concentrations greatly in excess of those that inhibit COX. Therefore, COX inhibition alone is not toxic.[The next sentences propose mechanisms for the toxicity of these agents and relate directly back to the reason for performing the experiment.] APAF arrests most cells in late G1 and S and produces a mixed form of cell death with both oncosis (swollen cells and nuclei) and apoptosis. APAF is known to inhibit the synthesis of DNA and cause chromosomal aberrations due to inhibition of ribonucleotide reductase. Such effects of APAF might account for renal medullary cell death in vivo and development of uroepithelial tumors from surviving cells that have chromosomal aberrations.

Introduction [describes the background and the purpose of the experiment.This will probably be the longest part of your paper. You will need to describe your substance: what it was, why you chose it, how it was used, any questions regarding its use. You also need to discuss your model, i.e. the type of cell you used: why this particular model or tissue as opposed to another, the usefulness of assessing the effects of your substance on this model, what previous studies have been done using either the model or the substance. You may have difficulties finding information about your substance. Most, if not all, of this information requires referencing. I will assist you; let me know if you're having difficulties! State exactly what you want to know about your substance. You may want to include a figure in your Introduction to support your explanation of why you chose a particular substance. If you use an image from a website, you must cite the website in the figure legend and in your Literature Cited section. Failure to do so constitutes plagiarism.]

Combinations of nonsteroidal antiinflammatory drugs (NSAIDs), taken in large doses over a prolonged period, cause a specific form of kidney disease, characterized by papillary necrosis and interstitial scarring (1). [a brief description of the substance under investigation and problems with use of the substance] [Numbers in parenthesis indicate references where this information was obtained. Every journal will have a preference for how references are indicated. This journal used order of citation. Others may use alphabetical listing. All references are cited within the text of the paper. No reference is included in the Lterature Cited section that is not cited in the text.] The patients have progressive chronic renal failure and are susceptible to the subsequent development of uroepithelial tumors. It is not clear what the mechanism of the drug toxicity is, but there are many theories, including the effects of cyclooxygenase (COX) inhibition, direct toxicity owing to elevated concentration of the drugs in the medulla, anoxia, and metabolic effects (2, 3). Epidemiological evidence incriminated mixtures of drugs including aspirin (ASA), and phenacetin or acetaminophen (APAF), the active metabolite of phenacetin (4). The mixtures often also contained caffeine. Interestingly, in some epidemiological studies caffeine content was the best predictor for subsequent renal failure (5, 6). The renal medullary toxicity of these particular analgesic drugs was apparently supported by animal studies, but interpretation of both the epidemiological and animal studies has been questioned (7-9). [Notice there are no quotations; everything is paraphrased. Scientific writing seldom, and I do mean seldom, uses quotations.]

There is also considerable evidence that NSAIDs slow the growth of tumor cells and cause them to die by apoptosis (10, 11). Those actions involve the inhibition of COX in some cases, but not in others. Furthermore, caffeine influences many pathways involved in the cellular response to DNA damage, reducing the cell cycle delay caused by DNA damage and inhibiting repair of the damage (12).

[Notice they specifically stated their hypotheses and how they proposed to examine these questions.] We hypothesized (i) that the NSAIDs and caffeine, which were implicated in renal medullary necrosis, may be directly toxic to renal inner medullary cells, causing apoptosis; (ii) that the toxicity could be enhanced by combinations of the drugs; and (iii) that the toxicity might be related to the inhibition of COX. To test these hypotheses we exposed renal inner medullary collecting duct (mIMCD3) cells that were rapidly proliferating or confluent to salicylic acid (SA), ASA, APAF, NS398, indomethacin, and caffeine, either individually or in combinations, and examined the effects on cell number, cell cycle, and cell death. We find evidence of direct toxicity of all of these drugs. Toxicity is particularly evident with the combination of caffeine and APAF. We also found that COX inhibition per se is not sufficient to produce these effects and that APAF kills the cells by oncosis as well as apoptosis. [The term oncosis (derived from onkos, meaning swelling) was proposed in 1910 by Von Recklinghausen precisely to mean cell death with swelling. Oncosis leads to necrosis with karyolysis and stands in contrast to apoptosis, which leads to necrosis with karyorhexis and cell shrinkage (13, 14).] Furthermore, toxicity is considerably greater in rapidly proliferating cells than in more slowly growing, confluent cells.

Materials and Methods [a description of how the experiment was done. This section should contain sufficient detail to allow another experimenter to repeat the work. Use the past tense; do not include any results or discussion. You are not writing a "cookbook". Do not copy the procedures described in the course website. Since most of these procedures are in standard usage in biology, merely summarize what method was used. See the example below*. The following is an abbreviation of the Materials and Methods in the paper.]

Cell Culture. mIMCD-3 cells (15), generously provided by S. Gullans (Harvard Medical School), were used in passages 14 to 20. They were grown in medium containing 45% DMEM (low glucose), 45% Conn's improved medium mF12 (Irvine Scientific), 10% FBS, and L-glutamine (2 mM) (Life Technologies, Rockville, MD) at 37°C with 5% CO2 plus 95% air. [You will state what kind of cells you used, for example, cardiac myocytes from a 7 day embryonated chicken or a cancer cell line, the type of medium you used, and the environmental conditions used for maintaining the cells.]

Drugs. APAF (4-acetoaminophenol, lot 107H0332), acetylSA (lot 46H105325), caffeine (lot 77H01221), SA (lot 48H0206), and indomethacin (lot 38H0906) were from Sigma. NS398 (lot 12923p) was from Cayman (Ann Arbor, MI). All drugs were directly dissolved in the medium except indomethacin and NS398, which were dissolved in DMSO and then diluted with medium to a final DMSO concentration of 0.1% or less. The same concentration of DMSO was added to control cells, as appropriate. [What substance did you use? Was it dissolved in water, in the feeding medium or did it require dissolving in another substance prior to addition to the feeding medium? What was your control?]

Experimental Design. To study cells in the log phase of growth (rapidly proliferating cells), 1,000 cells were plated for 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay, or 2,000 cells were plated for laser scanning cytometry (LSC) assay, then after 8 h for attachment, the control medium was replaced by an otherwise identical one, containing one or more drugs. The media were renewed 2 days after the drugs were first added, and the cells were analyzed after 3-4 days. Experiments were performed in the presence of 10% FBS. [Did you count your cells? When did you add your substance? How long were your cells exposed to the substance? How did you assess whether or not your substance was toxic to cells?]

The MTT assay was used to estimate cell number as previously described (16, 17). [*Notice that the authors reviewed briefly what they did and referred the reader to another article for further details. Don't rewrite the manual here!] Briefly, mIMCD3 cells were seeded on 96-well plates (Costar 3596) and were allowed 8 h to attach. Then, control or experimental media were substituted and remained for the time required by the experiment. At the termination of the experiment all media were replaced with serum-free control medium, containing MTT, 1 mg/ml, for 2 h at 37°C. After the MTT solution was aspirated, the blue formazan reaction product was extracted from the cells with propanol for 10 min with shaking, then the optical density was read on a Labsystems Multiskan MCC/340 microplate scanning spectrophotometer at 540 nm with background subtraction at 690 nm. The result for each condition in a single experiment was determined as the mean of measurements of eight separate wells.

Results [describes the observations and results of the experiment. This section will refer the reader to tables, graphs and/or figures. Data and observations are presented clearly and succinctly. Avoid presenting the same data in more than one way.You should have at least one figure or table in this section.]

Four days of exposure to ASA, SA, APAF, or caffeine greatly reduces the number of rapidly proliferating mIMCD-3 cells, as measured by LSC (Fig. 1A) or MTT (Fig. 1B). ASA, SA, or APAF at a concentration of 0.5 mM reduces the cell number to 40-80% of control, and 2.0 mM reduces the cell number to 20% of control or less. Caffeine is less effective, having little effect up to 1.0 mM.

Fig. 1. Effect of APAF, ASA, SA, caffeine, or APAF plus 1 mM caffeine on the number of rapidly proliferating mIMCD3 cells, measured by LSC (A) or MTT (B). The cells were exposed to the drugs for 3-4 days. Each point represents the mean of two to four (A) or two to six (B) independent experiments at each concentration. [The results of an experiment can be depicted in a visual format using graphs or figures, or as a numerical or written record of data in a table. Graphs and figures should always contain a descriptive legend, not usually a complete sentence, that adequately explains what is being depicting without reference to the text of the paper. Notice that the authors do not merely state what is on the labeled axes. Graphs are considered figures and should be designated as Fig. #. Tables should also have a brief descriptive title. If you use more than one figure or table in your paper, number them in the order they appear in the text of the paper. Number the figures independent of the numbering of tables. Always refer the reader to the figure or table where appropriate in the paper. Remember: figures and graphs should be simple, accurate and clearly understood. Your figures will probably not be this detailed; you are assessing the effect of one substance; they tested four substances at 5 different concentrations. This type of experiment is called a dose response.]

We also attempted to determine whether the drugs might be more toxic in combination than singly. In screening experiments we tested the combinations at several different concentrations of each drug. In general, the combinations are at least partially additive, as demonstrated by the example in Fig. 2A, which is not surprising. Of special interest, however, one combination, that of APAF plus caffeine, has a particularly large synergistic effect (Figs. 1A and B and 2A).

Fig. 2. (A) Effects of combinations of 0.1 mM SA, 0.5 mM APAF, and/or 1.0 mM caffeine for 4 days on rapidly proliferating mIMCD3. The cell number was measured by MTT after 4 days of exposure to the drugs. (B) Effects of SA, APAF, and caffeine on confluent mIMCD3 cells. The concentration of each drug was 2.0 mM. After 4 days of exposure to the drugs, the cell number was measured by MTT (mean ± SEM, n = 4, *, P < 0.05 versus APAF alone). FBS was present during the experimental period in two experiments, but not in the other two. Because the results do not differ, they are combined. [You may want to present your data as a bar graph such as this figure. The y-axis is the same as in Fig. 1 but the x-axis shows combinations of the drugs at fixed dosages. You may design your experiment as a dose response or as a fixed dosage.]

Discussion [an interpretation of the results of the experiment and an analysis of how these results support or refute previous studies. Discuss if you saw what you predicted in your hypothesis or if your results deviated from your predictions. Point out any flaws or inadequacies in the design of your experiment. Suggest what you would do to rectify problems. What did you learn by this study?]

NSAIDs and Caffeine Are Directly Toxic to IMCD Cells in Culture. Our results demonstrate that the NSAIDs ASA, SA, APAF, indomethacin, and NS-398 are directly toxic to mIMCD3 cells in tissue culture, as is caffeine. Excessive ingestion of NSAIDs and caffeine has been associated with chronic renal failure (1). In what follows we discuss the possibility that these drugs might directly damage renal medullary cells in vivo, contributing to analgesic-associated renal disease. We will consider (i) the role of COX inhibition in the observed effects, (ii) comparison of the drug concentrations that are directly toxic in tissue culture with concentrations that might occur in renal medullas in vivo, and (iii) possible mechanisms of toxicity. We conclude that direct toxicity to renal medullary cells in vivo is a plausible, if unproved, factor contributing to the renal toxicity of NSAIDs taken in excess and that the small population of renal inner medullary cells that are proliferating may be particularly vulnerable, explaining why only very high doses of the drugs, taken over a very long time, lead to chronic renal failure. [discussion abbreviated]

References [all the references cited in the paper. Most of these citations will be in the introduction. Do not list a reference that was not cited in the paper. Include the authors last name, initials; any other authors listed last name first; the title of the reference; the name of the journal; the date and journal publication number. Check and recheck the accuracy of your citations. Provide the reader with all the information necessary to locate a source. Internet sources are not acceptable in scientific papers. However, in this course, I will accept internet sources only if you review them with me prior to handing in your paper. I require the website address submitted to me in writing; give me 24 hours to determine if it is acceptable. If something appears here that I have not approved, I will deduct points from your grade on this paper.]

1. No authors listed. (1984) Analgesic-associated kidney disease, J. Am. Med. Assoc. 251, 3123-3125. [PNAS does not require the title of a paper to be stated in the reference section. I do. I added the title of this first reference as an example.]
2. Shelley, J. H. (1978) Kidney Int. 13, 15-26.
3. Zambraski, E. J. (1995) Semin. Nephrol. 15, 205-213.
4. Klag, M. J. , Whelton, P. K. & Perneger, T. V. (1996) Curr. Opin. Nephrol. Hypertens. 5, 236-241.
5. Pommer, W. , Bronder, E. , Greiser, E. , Helmert, U. , Jesdinsky, H. J. , Klimpel, A. , Borner, K. & Molzahn, M. (1989) Am. J. Nephrol. 9, 403-412.
[list continues]

Further Suggestions for Writing

1. Know your audience! Write your paper at the level of BIO 103. Explain or define terms or phrases that may be unfamiliar to your audience. Always remember: If you had to look it up, your audience probably will not understand it either.

2. Watch that sloppy writing! I am not here to teach you basic grammar and spelling. I will quit reading your paper if I have to spend my valuable time and energy serving as your editor. If you are having difficulty with your writing (usually with organization), let me know and I'll help you. I will check your final paper for the following: awkward phrases, spelling errors, evidence for lack of proofreading, inappropriate verb tense, inappropriate subject and verb agreement, problems with punctuation, continuity and structure, unnecessary material, completeness, consistency, coherent paragraphs.

3. Never turn in a first draft! Although I have asked you to submit a draft, it should not be your first draft. Edit, re-write and then I will help you fine tune.

4. Use the stylebooks available in the library.

5. Use an outline to organize your ideas and writing.

6. Be careful of your references. Do not include any references in your Literature Cited section that are not directly cited in the text of your paper. Failure to cite references is considered plagiarism. Use an appropriate and consistent citation style. I need to approve any internet sites that you use as a reference. Use the following format when citing these web sites:

Plagiarism: What It is and How to Recognize and Avoid It (Last updated: April 17, 1998) [Electronic version]. Retrieved April 30, 2004, from http://www.indiana.edu/~wts/wts/plagiarism.html