*Someday soon I’ll have time to write again!
“Money won is twice as sweet as money earned.” The Color of Money
Teaching has kept me busy since my last post. The days leading up to midterms, the grading and the office visits by concerned students in the aftermath have been too full for blog writing. In my biochemistry lecture course, the students and I are making our way through the core metabolic pathways, and I’m trying to come up with creative ways of getting the main ideas across.
First stop: Glycolysis Blackjack
Glycolysis is a universal metabolic pathway for all organisms that consume glucose. (Yes, that includes plants. They just happen to make their own glucose from sunlight and CO2 instead of eating other organisms.) As far as energy-yielding pathways go, it’s not that complex. Glucose molecules are converted to pyruvate yielding a net of 2 ATP molecules. However, the names of the enzymes and the metabolic intermediates all start to sound the same and it’s easy to get lost in the details. Here’s an analogy to keep the overall picture in mind.
ATP is often referred to as the biochemical cash of the cell. The simplest game to win some ATP is at the glycolysis black jack table.
First, you have to pay to play. Invest an ATP to get your cards.
Congratulations! Glucose-6-Phosphate is just like being dealt a pair of aces.
If you’re dealt this hand at any casino biochemistry or otherwise, your next move is to split those cards into two hands. (You’ve either got 2 or 12 and the chances of you beating the dealer are much better if you know you are starting with an ace.) As in any casino, you have to pay to split; so you invest another ATP. In glycolysis, you’re splitting a 6 carbon sugar into 2 3-carbon molecules (glyceraldehyde 3-phosphate).
In this contrived situation at the biochemistry casino, betting is limited to your investment. However, when you get your second card for both of your hands, you get jacks. Black jack on both hands. You win! Your winnings on one hand mean you break-even on your investment. Your winnings on the other hand mean you net back your initial investment (2 ATP). In glycolysis, the two molecules of glyceraldehyde 3-phosphate can each be used to yield 2 ATP molecules, giving a net of 2 ATP from glucose.
Sure, it’s not the drama of winning at slots, roulette or the lottery. But at this casino, you always get these cards. If you can ante in the first ATP molecule, you’ll get a pair of aces. If you can ante in the second ATP, you can split them and get jacks and double your investment. Every. Time. Sure, betting is limited, but if you’re guaranteed to win, you would sit at that table all day and all night. And you do. Eventually, you take those winnings and those cards to another table with higher stakes, but that will be a separate analogy. Other organisms make a perfectly good living at this table alone using glycolysis coupled with fermentation.
Of course, there isn’t a money casino in the world that works this way. The house always wins. But you should really split those aces when you get them. That’s still good advice.
Here’s a link to the BiochemistryBlackJack Powerpoint slide with animation if you’re interested in using the analogy.
A special thanks to my husband SuperChef for patiently explaining the finer details of blackjack to me to make this analogy work.
This is part of a series of tutorials I’m putting together for my students.
So, you have collected some interesting data from your experiments. Since no one but you will be reading your lab notebook (but hopefully people could if they wanted to), you need to present that data in figures so the rest of the world can know what you did and decipher your results.
Deciding what data gets to be a manuscript figure
The purpose of your manuscript is to show evidence for a new conclusion and the data presented in your figures should tell this story. Remember, the order of your figures for your manuscript may not necessarily (and probably won’t be) the order in which you collected the data. So, once you have all of your figures assembled, print them out one per page and work on defining the best order of presentation to make the case for your new conclusion. Now is a good time to evaluate whether there are any potential weaknesses regarding support for your conclusions, either in the data you already have or data you may still need to collect. You want to present the strongest possible case before your manuscript is submitted for review, but everything is a cost-benefit analysis and you’re always against the clock.
At this point you may also notice that some of the data presented in figures may be tangential, not quite fit with the rest or break up the flow of the story you are trying to tell. Authors can decide to cull certain figures (Sorry, data you have to remain in the lab notebook) or move them to ‘Supplementary Figures.’ Many journals allow for the inclusion of Supplementary Material- extra figures, longer versions of methods, large tables of data or files that would never be appropriate for print format. These Supplementary Materials exist as electronic files only linked to your final accepted manuscript as it appears as a journal article. Each journal has a different policy for what is acceptable for Supplementary Material. Some are more inclusive- the more data the merrier, drag everything out of all authors’ lab notebooks. Others are very limiting- essential data and files in appropriate for print only and anything else must be incorporated into the main body of the manuscript or cut out completely.
Preparing figures starts with high-quality data.
Images should be of sufficient resolution. Any adjustments of brightness and contrast must be made to the entire image; adjusting selective portions is unethical data manipulation and scientific fraud. Cropping is OK, but again beware of excessive image manipulations. They are usually an indicator that you need to repeat the experiment to obtain the necessary data.
Experimental data should be free of technical errors or other artifacts. The results should come from experiments as described in the methods section. Consistency in following experimental protocols (and including all of those details in your notebook) should be standard lab practices. Controls must be performed for each experiment so that the results can be properly interpreted. As you evaluate the figures you have made from your data, check again to see if all necessary controls have been included. When in doubt, don’t skimp on this- repeat the experiment with the proper controls. Your co-authors and reviewers will likely eventually tell you the same thing.
Your data should also be repeated enough times to be statistically relevant. Note that this does not mean you repeat an experiment enough times until you get the data you want. This ‘cherry-picking’ is another unethical manipulation of data. Unfortunately, this type of fraud is the most difficult to catch by the peer-review system. Reviewers have no way of knowing that you have a hundred other experimental trials with contradictory data in your lab notebook. Scientists must have the integrity to accurately present their results and have legitimate justifications for excluding some data (altered variables, confounding variables, improper controls etc). It is not always possible to show all repetitions of an experiment and in some cases (like gels) it is not even feasible to average the results. Showing ‘representative data’ (a single instance of the most common result) is perfectly acceptable, but it should be just that- representative of your average results.
Don’t pursue perfect data at the expense of integrity. The rising standards of scientific work and competition for rewards based on that work create an enormous amount of pressure to compromise your integrity for the sake of publication. RESIST! Research fraud undermines our entire enterprise. Biological systems are inherently complex and imperfect- we should not expect results to be simple and pristine. Control for what you can when you can, but do not otherwise force data to yield a certain result.
Putting together figure files
Usually your data will consist of images or graphs. These electronic files must be edited to include the raw data as well as appropriate labels. The simplest way of doing this is to drop the images into a PowerPoint slide to assemble all the necessary parts. Text boxes can be used to add labels. Lines and arrows can be added to draw attention to certain features. All labels and features of your figures should be properly aligned using the automatic tools for doing so. More complex figures consist of multiple parts that are designated by letters (Ex: Figure 1A and Figure 1B), and these letters can be added as text boxes. Journals tend to have preferences for the exact labeling details (fonts, sizes etc) and the instructions to authors will have this information. Make sure you read this information carefully and apply it consistently across all figures. Don’t use Arial capital letters to label the parts of Figure 1, Calibri Roman numerals for Figure 3 and lower case Times New Roman on Figure 5. You’re not in cloud cuckoo land. Editors, reviewers and other scientists appreciate consistency.
Remember that in the final manuscript format, the sizes of all labels and images will be considerably reduced. Make sure that your figure as submitted in manuscript form is sufficiently large so that it is still interpretable at a much smaller size. Any lines on graphs should be of sufficient thickness so as not to disappear or lose their pattern upon reduction. Note that it is generally easier to number samples like gel lanes, mulitpart images, etc than to write out the full sample description in the figure. Save the full sample names and descriptions for the figure legend.
When available, move up in the food chain to a program like Adobe Photoshop or Illustrator or Corel Draw to put together figure images. These programs have a steeper learning curve, but offer more sophisticated options for putting the figure file together and saving it as a high resolution image. For many journals, your figures must be submitted as image files (usually .tiff) or as PDF pages. Most journals use the manuscript submission phase as their quality control phase, meaning the files you submit for review must be of sufficient quality for the manuscript proof. Speaking of higher quality software, programs like OriginLab and Kaleidagraph are much better at generating image quality graphs than Microsoft Excel.
Color vs. Black/White or Grayscale Figures
Journals will typically charge you more to print color figures over black and white or grayscale images. (Oh, so yeah, if you didn’t get the memo, the authors typically pay publication charges to cover the printing and/or access for the published work. But then again, if you’ve gotten this far, you’ve realized you’re not in science for the money.) When possible use black and white or grayscale figures. If graphs become too complicated in monotone, try breaking up the number of samples shown on the same axis. Of course, you shouldn’t completely eschew color. Use it when it is most appropriate to distinguish samples. For example, it’s not that big of a deal to show a Coomassie-stained gel in black and white, but pictures of Arabidopsis showing wild-type and mutant plants with varying degrees of pigmentation should definitely be in color. Finally, as part of the ‘use color judiciously rule’, stick with the basic colors (8 crayola box, not the 196) so that there are no incompatibilities or unexpected shifts in tone when transferring files or changing file-types. Also keep in mind that there is ~10% prevalence of red-green colorblindness, so avoid using these colors together to differentiate between key samples. (Hey, after #TheDress this week, maybe you should just avoid color altogether.)
The figure heading, title and legend
Each figure should have a heading as defined by the journal (Ex: ‘Figure 1.’ ‘Fig. 1’ or ‘Figure 1:’). Each figure should also have a title, the formatting of which may be explicitly defined by the journal. It may be required to be in the form of a complete sentence or just a concise phrase; it may be required to be in bold or italics to distinguish it from the legend. The legend should tell the reader what they are looking at. It is not necessary to include lengthy procedural details, but it is useful to mention the name of the experiment and any details about treatment or sample preparation useful for interpreting the data in the figure. It should define all parts shown. Every sample or label on the figure must be defined in the legend.
Other random and lesser commandments
Include your figure and figure legend tips and lesser commandments below.
This is part of a tutorial series I’m writing for my students. Science writing first requires a lot of science reading.
Any scientific manuscript will require numerous other references to scientific literature to substantiate the facts upon which it builds. This means you have to become familiar with a body of literature related to the topic. Finding reliable references and sorting out what they mean is no small task. As a scientist, it is useful to make literature searching and reading a regular part of your routine. Set a goal to read a certain number of papers each week to keep up with the research in your area. When you are in ‘writing-mode’ for a grant or a scientific manuscript, the reading will likely be more intense, but it is a general good practice to keep up with the scientific literature a little bit at a time.
Searching for Literature
My go-to search engines for finding scientific literature are Google Scholar and PubMed. You can search key words, titles, authors, year, narrow by article type (review, patent, clinical study, research article etc). There are also numerous options for narrowing down your search and sorting the results (relevance, dates etc). The links for the titles can generally take you to the full text of the article (beware of paywalls if you’re not on a network with an institutional subscription). If based on the abstract of the article, you’d like to get a look at the full text, but you’re on the wrong side of a paywall, you could always e-mail the corresponding author to request a copy. For a quicker response, tweet the reference with the hashtag #ICanHazPDF with email [at] domain.com and someone out there on the interwebz with access will send it to you. One of the biggest issues you will likely have is sifting through the long list of titles for something that is actually useful to you. When embarking on a new literature search, try to find a recent review article to give you an overview of the topic and point you to relevant primary research articles. Then just start reading and following citations through the literature until you have all of the information you need.
Reading Primary Research Literature
The best way to understand scientific papers is to practice reading them. There’s a learning curve for the jargon and background in your field, but sometimes papers are also just crappily written. By nature, scientific literature is information dense and since it continually builds on previous studies, the reader always enters in the middle of the action. If the paper is well-written, the abstract should give you a sense of the importance of the work, the research that was done and what it means. The introduction should give you just enough information to allow you to understand the research question that will be addressed. In primary research papers, the introductions should be fairly focused. Consult a review article for a broader scope of what’s going on in any given research area. Unless you are looking for a particular protocol or are trying to replicate an experiment, the methods section probably won’t get much attention reading through an article for the first time. The first time through, just get an idea of the techniques used in the research, but wait to sort out any of the finer details until you see the figures in the results section. As you’re sorting through the results section, this is where the authors are showing you their data. They should explain some of the rationale behind the line of experiments, what data they collected and what it means. There should be some connection or flow among the figures and results that ultimately builds up to (an) overall conclusion(s). In some articles, the data are presented to build a case for a certain model or overall conclusion. In others, the experiments are geared towards eliminating possibilities until the results focus in on a particular conclusion. Many papers aren’t always written this way and there is a greater burden on the reader to interpret the results to see if it leads you to the same conclusion. Personally, I like papers that can end with some sort of model (cartoons are appreciated) to visually sum up all of the conclusions. In any case, the reader should give a critical eye to all results in the relation to how they are supporting the conclusions. Look at the figures and write down your own results; are they the same as the description the authors give? Ask yourself if the results mean what the authors say they mean. Could there be any other possible interpretation of the results? Then think deeper about the data they are showing. Are those experiments the best way of figuring out what’s going on? Is the data of sufficient quality (error bars, statistics, clarity in images etc)? Did they perform all of the appropriate controls? Refer back to the methods section for finer points of the protocols. Are there any red flags about how the work was performed that could influence the results? The peer-review process is not perfect, so even though it is science’s way of validating work prior to publication, it doesn’t mean that there are no mistakes or misinterpretations. After all, the science was performed, written and evaluated by humans.
Keeping up with the Literature
There are a few ways to make sure you are keeping up with the literature in your field of interest. Set aside some time each day or week to at least scan the titles of what’s going on in your research area. Set a goal for paper-reading, even starting out at 1 paper/week will be useful. Check out the hashtag #365papers on Twitter for inspiration and accountability. There are a few different ways to automate the process as well. Sign up for eTOC (electronic table of contents) alerts from journals you read often. The contents of each issue will be delivered to your inbox as soon as they are available. Give them a quick scan for interesting key words and download what seems interesting or relevant. Harness the power of the Google search engine and sign up for Google Scholar alerts. Based on keywords that you provide, Google Scholar will send you daily updates with relevant literature.
As a scientist, you will begin to amass numerous downloaded PDF files of research articles. It’s your own digital library for the full-text version of useful papers. For many, this collection is purely electronic. Gone are the days of file cabinets filled to the brim with reprints and papers with scribbled notations in the margins. If you prefer, printing and hand-writing notes is still possible, but not feasible for every paper you will ever read. So, you need to come up with an organization system that works for you. I have a folder where I dump all of my papers and they all have filenames (year first author name). It’s rudimentary, but it works for me. I can often remember which paper I need to open, but when I can’t it’s an easy search. Writing this tutorial has shown me that I’m still in the dark ages with respect to my organization strategy. There are a number of software options for helping you manage your own digital library. Check out Papers, Mendely, CiteULike. These programs offer more options for searching and sorting your own digital library as well as some integration with internet-wide literature searching.
Give your favorite searching, reading and organization tips in the comments below. Please weigh in on a literature organization/management software preference because I’m embarrassed to have not gotten with one of these programs yet. #OldSchool #AtLeastItsNotPaper
This is part of a tutorial series I’m writing for my students on scientific manuscript preparation.
This section should include information on every experimental procedure used in the work. You don’t have to include experimental details of everything that was in your lab notebook or all of the optimization work you did for the project. Yeah, readers don’t care about the two-years’ worth of failed protein purification methods in your notebook. You will only include the short paragraph of what did finally work. Refer to your figures. Whatever experiments yielded data that is ultimately found in your figures should have details in your Materials and Methods section. If certain reagents were critical to completion of the experiments, then include your sources for them (commercial or donations from other labs). The Materials and Methods section is written in the past tense without the use of personal pronouns. It may seem awkward to get into this point of view, but there is an omniscient perspective from which your methods can be written. I’m not sure where this convention originated, but remove yourself from the experiment and it seems more legitimate.
Subdivide this section into useful parts. This kind of organization also helps the reader pinpoint details for replication or evaluation of experiments. Often readers will be looking at your work to perform similar experiments or repeat your work.
It’s not exactly a protocol, but it should be complete. You don’t have to tell your readers every step of a protocol, but it should be complete enough for someone else with comparable training to replicate. The literature varies widely in the usefulness and clarity of methods descriptions. For more complex procedures, authors are often contacted directly for lab protocols or additional information not included in the materials and methods section. As methods have become more complex, fewer details are included. This is especially true of molecular biology work. Typically primer sequences are given and specific vectors are mentioned, but it can be especially irksome to figure out others’ cloning construction strategies. Usually, though, this information can be obtained directly from the authors. In general, I would err on the side of completeness (vs. concise) because this makes it easier for your experimental strategies to be evaluated and replicated. This strategy can run afoul of the journal’s character limits, but in this age of online publication, supplemental information is allowed. You can write a ‘Supplemental Methods’ section which will be available online only and can be as long as you’d like.
Referencing previous literature is a convenient way of being concise. It is acceptable to say something like, “This experiment was performed as in Reference (1).” However, if you go this route, make sure that the reference you are citing in this case does have the necessary information on experimental details. The scientific literature is infamous for pulling this trick only to reference yet another paper that says “This experiment was performed as in Reference (2).” You may have to follow this rabbit hole back a couple of decades before you find any real useful information on performing the experiment. (This is a personal pet peeve of mine that just makes me feel stabby when I encounter this nonsense.) So save your readers some trouble and either include the necessary info or cite a direct source. For many labs, there are standard references and descriptions for routinely used methods that appear in almost all of the manuscripts. It’s fine to revert back to previously published articles to see how things were worded and what references were cited, but it’s always a good idea to verify these references yourself. Otherwise, a mis-referenced citation will be propagated many times over. (This is also a good practice for any citation anywhere in a manuscript- to make sure the reference is really saying what you think it does.)
Offer your pro-tips for methods-writing in the comments section below.
This is the next post in a series of tutorials I’m writing for my students related to scientific manuscripts.
For every statement of fact in your introduction, results or conclusions sections or for every method, it is important to cite the appropriate reference, which provides the basis for this fact or method resource. Thus, references are given throughout the manuscript to direct the reader to previous literature to independently verify the facts or otherwise track down more information. Providing references for information in a scientific manuscript is somewhat different than providing references in a research paper for the humanities. While there may be some similarities with MLA style, there are specific requirements for scientific manuscripts that are determined by the choice of journal for submission.
Acceptable references are books and other peer-reviewed articles. You’re not allowed to get facts from random sites on the internet and other unreliable sources. Scientific literature is vetted by a peer-review process, in which other scientists evaluate the merit of submitted work. While it’s not perfect, this arduous process of evaluation and revision is science’s way of validating reports of experimental work. It means you can trust the information within peer-reviewed journal articles more than you can the random blog post or Wikipedia entry.
Specific formatting for references can be found in the instructions for authors. Some journals prefer using numbers in parentheses, some use superscripts, and some use an (author, year) formatting within the text of the manuscript. Reference lists are likewise variable according to journal- numbered lists, alphabetical lists, include titles, no titles, full journal names, abbreviated journal names etc. etc. In any case, the format will include enough basic information for a reader to look up the exact reference and check your facts. You have to follow the exact style for the journal of choice or the editor in charge of looking over new manuscript submissions will notice and kick your manuscript back to you- not reviewed, not accepted, not any further beyond your lab notebook than your computer files.
Citation management software should be your new best friend. Chances are the previous written assignments you’ve had haven’t required dozens of citations backing up various facts in your manuscript. You also probably worked on all of your previous manuscripts from start to finish in the order in which they will appear. As stated in my previous tutorial, you generally work on the sections of your scientific manuscript in a very different order than the one in which it will be finally assembled. Not to mention that working with multiple authors means merging sections from different writers and major rearrangements of written work. All of these complications mean that it is futile to try to manually keep track of all of the references that go along with these edits.This is a waste of your time and will lead to errors. Use citation management software. Citation management software like EndNote allows you to search for literature, manage all relevant information for a citation and integrate it into your handy text editing software (MS Word) in a way that lets you point-and-click which references to incorporate. This software will re-order citations in the reference list as sections are rearranged. The citation management software also already knows the exact details of each journal’s citation style. Simply select the appropriate journal option in the citation management software and EndNote will keep track of journal abbreviations, what should be italicized, what things are separated by commas and where periods should go. And if you decide to switch the journal for submission, all you have to do is click a different button and at least the reference section is done. Using citation management software ensures that no random numbers in parentheses are forgotten in the middle of your text.
Here’s a brief tutorial for using EndNote.
The EndNote software allows you to create a library of citations that can be used in your writing projects. There are two general strategies for EndNote library usage. (1) A single EndNote library containing every citation you will ever use. (2) Multiple EndNote libraries with one for each writing project. Each has its own merits and drawbacks. I know people that use both successfully, so if you feel strongly one way or the other, go with the one that seems most intuitive to you.
Citations can be added to your EndNote reference library by using the on-line search function. The most useful searches for scientific manuscripts are the PubMed (NLM) and Web of Science (ISI) searches. If you enter an author, publication year, journal name and some relevant keyword, EndNote should easily identify the necessary reference, which can then be saved in your library. It should be noted that I find this search feature useful for finding specific references (like those you’ve already read and know you want to use) rather than a general search to keep up with the literature (then again, I’m a type 2 EndNote user).
Reference entries can be added manually to an EndNote library also. If the paper is ancient or otherwise impossible to find with the search tools, you can manually type in all of the fields.
Duplicate entries within a library can be identified and eliminated. This should be done or it can cause problems when trying to use these entries while writing in MS Word.
The EndNote program also integrates itself as an add-on feature in MS Word. Once installed, it will have its own tab in the top toolbar. While working on a manuscript in MS Word, open the EndNote program and the appropriate library. From the EndNote tab in MS Word, all you have to do is click the ‘Insert Citation’ button to select the appropriate reference from the library. Note, the reference must be a part of the library at this point (already captured from an on-line search) because you can’t do an on-line search via EndNote from MS Word. Once selected, a reference placeholder will appear in the text (whether (#) or (author, date)) and a reference list will be started at the end of the text portion of your MS Word file. All of the details are determined by the designated style, given in terms of the journal title, which can also be changed from a drop-down style menu in the EndNote tab in MS Word.
Finer points of citation formatting with EndNote…
You can make some changes to the style if it’s not exactly correct based on the most up to date info on the journal’s website.
If you are citing multiple references at the end of a particular sentence, you can reorder them according to the stipulations of the journal style (usually in chronological order, oldest to most recent).
An EndNote library can be exported from an MS Word file. So, if you get a written portion from another author, you can recreate their EndNote library of citations even if they didn’t send you their library file.
A note on plagiarism: Don’t do it. Just because you are citing a reference in order to substantiate a statement in your manuscript doesn’t mean that you can copy their exact words. Use your own words and incorporate their work as it relates to yours. Don’t sweat whether minor wording changes are enough. Since you are building on these previous facts, the context will be different than the original paper, but there’s no excuse for copying things word for word. Sure, there are limited numbers of ways of saying any given truth clearly and concisely, but you can find a way of stating a necessary fact in your own words. Plus, you should really understand the previous literature well enough to restate necessary information in the context of your research manuscript without stealing someone else’s words.
Share your reference, citation and EndNote pro-tips in the comments section below.