How to read a scientific paper

Types of scientific information Primary source The first time any of the knowledge contained in the paper is revealed in the scientific community Secondary source Books, chapters, review articles Less specific, meant to be a summary of the current knowledge on the subject Tertiary source Almanacs, guide books, encyclopedias, textbooks Grey literature

Types of scientific information Primary source Pros: Up-to-date, reproducible Cons: Meant for experts, difficult to understand, may be expensive Secondary source Pros: Meant for informed beginners Cons: Can be out of date and may not completely cover all the knowledge on the topic, may be expensive Tertiary source Pros: Meant for beginners Cons: Can be out of date and incomplete Grey literature Pros: Free usually Cons: Little to no interpretation of the data

Types of scientific information Primary literature Carter GC, Bernstone L, et al. HIV entry into macrophages is dependent on lipid rafts. Virology 386, 192-202. 2009. Secondary literature Michalak E, Villunger A, et al. Death squads enlisted by the tumour suppressor p53. Biochemical and Biophysical Research Communications 331, 786-798. 2005. Tertiary literature Your textbook Grey literature 2011 World Malaria Report from the WHO

Original research articles and reviews Most advanced science information will come from primary or secondary sources Journals like Science and Cell will often have a mix of editorials, primary research, and secondary literature reviews

How to find primary and secondary sources Most English-language journal articles are indexed in a public database called PudMed http://www.ncbi.nlm.nih.gov/pubmed It will (almost) always have the abstract, a list of the databases the full text can be found in, and if you re lucky, a link to the free full text For example: http://www.ncbi.nlm.nih.gov/pubmed/21875946

Sections of a scientific paper Abstract Introduction Methods Results Discussion

From: http://www.lib.purdue.edu/phys/assets/scipapertutorial.swf

Abstract Summary of the paper Sets out the specific questions to be addressed in the paper and states the answers the authors found Short and sweet Can be read independent of the rest of the information in the paper

Introduction Explains, why bother? Explains prior research or knowledge necessary to understand the article Assumes a certain level of knowledge Defines the terms and abbreviations used throughout the article (Sometimes the definitions are in the abstract or materials and methods)

Materials and Methods Often the most dense/difficult part as you will not be familiar with most biology methods yet A well-written M&M allows you to exactly replicate the original experiment Contains the exact names, amounts, and manufacturers of the various components in the experiment

Results Contains all the data from the experiment The figures and tables contain the bulk of the information, with text explaining the most important bits Graphs, tables, pictures, etc.

Discussion Summarizes the experiment and explains the purpose of each figure Draws conclusions based on the results, and rewrites hypotheses as needed Reinterprets previous knowledge in light of the author s results Points towards areas of knowledge that still remain unknown

References Lists all the papers and works the authors cited Good place to look for further reading!

Tips on Reading Treat this like learning a second language. The more you practice, the easier it gets Be an active reader! Circle, highlight, write in the margins, re-read! Research words and terms you don t understand sooner rather than later Use all your available resources to research those unknown terms me, your book, the internet etc. Expect to spend 4+ hours per paper

What NOT to do Expect to understand everything the first (or even second) read-through Expect to understand everything, period Read everything in one sitting Not take notes and rewrite confusing sentences Do this the night before

An example paper Title: HIV entry into macrophages is dependent on intact lipid rafts Authors: Gemma C. Carter, Laura Bemstone, Dhaval Sangani, et al. Journal: Virology, 386 pages 192-202 Date: 2009 Hypothesis: HIV depends on the presence of specialized structures called lipid rafts in the cell membrane to infect macrophages

Background information Viruses (like HIV) infect cells by fusing with the plasma membrane Two types: enveloped and non-enveloped Enveloped have a lipid bilayer surrounding them Non-enveloped do not HIV uses a special enzyme called reverse transcriptase to convert its RNA genome into DNA during infection Lipid rafts Macrophages A type of white blood cell that helps combat infections

Receptor-mediated endocytosis and HIV Viruses infect our cells by attaching to cellsurface receptors meant for receptormediated endocytosis They fool the receptor by mimicking the natural protein that would usually bind HIV s gp120 protein binds to a white blood cell s CD4 receptor and CCR5 coreceptor Triggers endocytosis of the HIV virus Gp120 is a glycoprotein

Background information HIV infection in cells has three stages: HIV s gp120 binds to CD4 HIV then binds to CCR5/CXCR4 HIV fuses with the host s plasma membrane and enters the cell

Abstract

Understanding the introduction Highlight unfamiliar terms Research endosome Endosome = membranebound compartment that helps ships endocytosed material from the plasma membrane to the lysosome

Understanding the introduction Cholesterol is an important part of cell membranes that allows some viruses to fuse Cholesterol is concentrated in special lipid rafts along with other lipids Because of the presence of cholesterol in lipid rafts, it allows them to be separated from the regular cell membrane. They are then called detergent-resistant membranes (DRMs) Viruses may hijack the proteins embedded in these lipid rafts to enter The HIV protein Nef plays a role in raising and lowering cholesterol levels in the cell

Background information on how HIV infects cells

Understanding the introduction HIV needs its envelope to be infective Removing cholesterol from cell membranes using drugs prevents HIV infection CD4 is found with lipid rafts, but whether CD4 needs to be with lipid rafts for HIV to bind to it is disputed

GM1 = a type of lipid commonly found in lipid rafts

Understanding the introduction Whether CCR5 and CXCR4 (both coreceptors for CD4) are found in lipid rafts is disputed Possibly, HIV first binds to CD4 in a lipid rafts, then causes CCR5/CXCR4 to move into the raft so it can bind Another possibility: HIV is absorbed through a special type of pinocytosis Nef affects the function of a special transport protein called ATP-binding casette A1 Affects cholesterol transport

Identifying a gap in the current knowledge Hypothesis

Onto the results Figure 1A, 1B, 1C, 1D, 1E Figure 2A, 2B, 2C, 2D, 2D, 2F Figure 3A, skipping figs. 3B-3D Figure 4A, 4B, 4C, 4D, 4E, 4F Skipping all of Figure 5

Results Figure 1

Understanding the results figure 1 CTB-FITC is a stain that binds to the special lipid GM1 and shows where lipid rafts are on the cell membrane Shows that the virus clusters around CTB-FITC rich areas Does this directly shows that the virus clusters around lipid rafts? The virus most heavily clustered with CTB-FITC 20 minutes after virus and macrophages were mixed Other time points? This image contains both qualitative and quantitative information

Results Figure 2

Understanding the results Figure 2 The drug MβCD removes cholesterol from the cell membrane Figure 2A shows that MβCD disrupts lipid rafts in a dose-dependent manner What is the purpose of Figure 2A? Where is the control? Figure 2B shows that MβCD doesn t kill the macrophages What is the purpose of Figure 2B? Why is it necessary to show this?

Understanding the results Figure 2 Figure 2C shows that after treatment with MβCD, there were fewer reverse transcription products Where is the control? Is there dose-dependency? Does this directly prove HIV is not infecting the cell? Figure 2D shows that as soon as cholesterol was replenished, reverse transcription was restored by 43.5% Where is the control? What is the purpose of including 10uM MβCD? What is the purpose of including MβCD + Chol?

Understanding the results Figure 2 Figure 2E shows that the presence of p24 (a protein produced by HIV) decreases in the presence of MβCD Where is the control? Is there dose-dependency? What is the purpose of 10uM MβCD + 400ug/uL chol? Figure 2F shows p24 decreases in the presence of MβCD How is this different from 2E?

Results Figure 3A

Understanding the results Figure 3A Untreated macrophages expressed CD4, CCR5, and CXCR4 at detectable levels What is the purpose of untreated macrophages? Treatment with MβCD drastically lowered the presence of these receptors Replenishing cholesterol restores these receptors, except for CD4 MβCD does not lower the presence of all membrane receptors CD71, a non-raft receptor, remained at normal levels Would it be useful to measure the presence of other non-raft receptors?

Results Figure 4

DMSO is commonly used as a solvent for difficult-to-dissolve chemicals such as pharmaceuticals, proteins, etc. Results Figure 4

Results Figure 4

Understanding the results Figure 4 Nystatin and filipin both sequester cholesterol away from the cell membrane Figure 4A shows that both nystatin and filipin decrease CTB-FITC binding What does this mean for the lipid rafts? Figure 4B shows nystatin and filipin don t have a negative effect on cell growth at several concentrations Why is this necessary to prove?

Understanding the results Figure 4 Figures 4C and 4D show that nystatin and filipin decrease the presence of reverse transcriptase Does this directly prove HIV is not infecting the cells? Is there dose-dependence? Figure 4E shows decreased presence of p24 Why is it important to include both an untreated sample and a DMSO-containing sample? Figure 4F shows p24 levels after macrophage exposure to HIV for two hours Is there a control here that should have been included? How is this different from 4E?

Understanding the results Figure 4 Figures 4G shows the level of receptors in the presence of DMSO, nystatin, and filipin What is the purpose of CD71? Notice anything unusual about CD71 presence with nystatin? Does this throw off the integrity of the control? Is there consistency with nystatin/filipin suppressing receptor presence? Is it similar to the suppression pattern of MβCD?

Discussion Here, we have demonstrated the critical importance of cholesterol for entry of HIV-1 into macrophages. Firstly, we showed that productive virus entry is significantly reduced when the macrophage target cells are treated with MβCD, a cholesterol-depleting drug [ ] The addition of exogenous cholesterol to macrophage membranes directly after MβCD treatment substantially restored HIV infection, indicating that the decreased infectivity was at least part due to the depletion of cholesterol from the cell membrane. Secondly, we modified the properties of cholesterol-rich macrophage membranes using nystatin and filipin complex to sequester cholesterol into large aggregates. Treatment of macrophages with these drugs significantly inhibited productive HIV entry in a concentration-dependent manner. [ ]Therefore, modification of macrophage membrane cholesterol content using 4 different pharmacological inhibitors acting on cholesterol by different mechanisms all significantly inhibited HIV entry. Summary of the experiment and what was shown

Interestingly, CD4 expression was decreased further by cholesterol replenishment to almost undetectable levels. This complete complete knockdown in CD4 surface expression may explain why only a partial restoration of HIV reverse transcription was observed upon cholesterol replenishment of MβCD treated macrophages. The mechanism by which cholesterol further decreases CD4 surface expression is unknown, but as these cholesterol-treated macrophages have a more granular appearance, we can speculate that these macrophages actively take up cholesterol via an endocytic mechanism that may simultaneously internalize CD4 [ ] The reduction of CD4 surface expression upon MβCD and cholesterol treatment seems to be unique to macrophages. Tentative explanation of why the addition of cholesterol in Fig. 3A decreased rather than restored CD4 presence

Nystatin significantly decreases CD4 and CCR5 surface expression but also significantly decreased non-raft associated CD71, implying that the effect of nystatin may not be restricted to proteins located in cholesterol-rich domains Conversely, filipin complex treatment significantly reduced CD4 expression levels and slightly decreased CCR5 levels but did not appear to have such a dramatic effect on the expression of other membrane proteins The effects of cholesterol sequestration on the surface expression of the HIV receptors, after nystatin and filipin treatment, have not been investigated before. Tentative explanation of why nystatin decreased levels of the control CD71 in fig. 4G Outlining where these discoveries fit in current scientific knowledge

CD4 association with lipid rafts has been established in many different cell types and we can show this to be true for macrophage CD4. Here we show in macrophages, CCR5 is also present in or associated with lipid rafts. The most likely explanation for the inhibition of productive HIV entry into cholesterol-depleted macrophages is that redistribution of (or conformational changes to) the HIV receptors CD4, CCR5, and CXCR4 render the macrophages unsusceptible to HIV infection. It is plausible that manipulation of membrane cholesterol may inhibit entry by other mechanisms, of which we suggest five possibilities. 1) Depletion of macrophage cholesterol may alter the lipid composition.etc. Outlining where these discoveries fit in current scientific knowledge Stating the most likely explanation, but also identifying other competing hypotheses

The authors went through several steps to prove their hypothesis: Two measures of viral presence Showed that MβCD inhibited HIV entry through lipid rafts in a dose-dependent manner MβCD disrupted lipid rafts (Fig. 2A) MβCD had no effect on cell viability (Fig. 2B) MβCD presence decreased the presence of viral reverse transcriptase (Fig. 2C) MβCD presence decreased the presence of viral protein p24 (Figs. 2E, 2F) MβCD decreased the presence of CD4 and CCR5, suggesting a mechanism by which disruption of lipid rafts prevents HIV infection (Fig. 3A) Adding cholesterol back in after depleting it using MβCD restored (some) infectability (Fig. 2D)

Showed that both nystatin and filipin inhibited HIV entry through lipid rafts Nystatin/filipin disrupted lipid rafts (Fig. 4A) Nystatin/filipin had no effect on cell viability (Fig. 4B) Nystatin/filipin decreased the presence of reverse transcriptase (Figs. 4C and 4D) Nystatin/filipin decreased the presence of p24 (Figs. 4E and 4F)

Things to consider Why was it important that the authors include four cholesterol-disrupting drugs, all with different ways of removing cholesterol? What is the main limitation of this experiment? (Hint: cats on a tilt-a-whirl) Why is the knowledge gained in this experiment significant?

Extra Links Tips on reading a scientific paper http://www.biochem.arizona.edu/classes/bioc568/pa pers.htm Anatomy of a scientific paper http://www.lib.purdue.edu/phys/assets/scipapertuto rial.swf How to read a scientific paper http://www.sciencebuddies.org/science-fairprojects/top_sciencefair_how_to_read_a_scientific_paper.shtml#twotypes

Primary Source Review #1 Title: Age-associated decreased activities of mitochondrial electron transport chain complexes in heart and skeletal muscle: role of L-carnitine Authors: Kumaran S., Subathra M., Balu M., Panneerselvam C. Journal: Chemo-Biological Interactions Vol. 148 Pgs. 11-18 Date: 2004

Background information The decay of mitochondria is a major contributor to aging in three ways: Production of reactive oxygen species A side-effect of cellular respiration ROS = oxygen radicals = O 2 - Decline in the activity of the electron transport chain Decrease in the fatty acid composition of the mitochondrial membrane

Background information ROS damage macromolecules such as lipids and DNA ROS are produced in the mitochondria during cellular respiration The DNA sequences that encode for the proteins are present in both the nucleus and mitochondria

Background information The electron transport chain in mitochondria is a system of proteins that converts glucose into ATP There are four protein complexes in the ETC: I, II, III, and IV These complexes have several different components, such as FMN and FE-S for complex I

Questions?