Weekly Wrap-up

Just like what I told you at the very beginning of this class, for this journal club session, even I walked with you figure to figure, assay to assay, I guarantee you that you will probably forget everything in 2 or 3 weeks. Therefore, in this unconventional journal club session, I am going to just solve three questions, and three questions only: (1) Why to Read, (2) What to Read, and (3) How to read, an research article. 

Over the past four sessions, we've delved into why reading research articles now is a good idea. By doing so, you're essentially given a VIP backstage pass to the world of science, where you can dive deep into new discoveries and kindle your passion. We've also navigated through databases like Web of Science, PubMed, and Google Scholar using relevant keywords, weighing their pros and cons, and handpicking two papers for detailed study. I've also given you a taste of using these databases, like Web of Science, to discover the heavyweights in any research field. This way, you can quickly grasp a bird's eye view of any field you've started to fancy.

In the latter half of this week's sessions, we jointly demystified the structure of a research article - what's in the title, how to decode it, the basic layout of an abstract, and what each paragraph in the introduction/summary covers. I've also guided you on formulating hypotheses and designing experiments to test them, particularly the alternate hypotheses. For our next week's rendezvous, we'll plunge deeper into the details of Paper I and Paper II. I'll walk you through the storytelling and logic of the authors, the varied types and essential elements of figures/tables, and how to interpret and present a figure or a table.

Over this weekend, please take the time to read both Paper I and Paper II. Reading a research article for the first time can seem daunting, but fear not! Start with the introduction/summary section of Paper I, read it aloud, and then move on to the results. After each paragraph, pause and reflect on the background, conclusion, and evidence provided in that segment. You'll probably find that your thoughts align neatly with the structure of the paragraph, which is no coincidence - it's how research articles are crafted.

In our last session on Friday, we split the class into three groups, each tackling one figure. I'd like you to ponder over the following questions and write your answers down:

1. What is the research question? 

As we said in class, a research question is different from a hypothesis. A research question is an open question; while, a hypothesis is a statement.

2. What's the hypothesis?

A hypothesis is a firm sentence as a statement. Do not confuse yourself between research question and hypothesis.

3. How did the authors test their hypothesis?

What is the research method/techniques? What is the research material? Did they have set proper controls (MUST Have BOTH Positive and Negative Controls)? 

4. If the hypothesis is correct, what would the experimental results look like?

If the hypothesis stands up to testing - for example, the hypothesis that "Agro-infiltration can lead to high-level expression of spike proteins in tobacco leaves" - then the evidence will be clear in the experimental outcomes. Specifically, you'd expect to see a significant increase in spike protein concentration within the crude protein extract derived from the tobacco leaves. This higher abundance of spike protein would be a direct indication that the agro-infiltration method has been successful in overexpressing the spike protein, thereby supporting the hypothesis.

5. What other POSSIBLE outcomes could the experiments have?

Think of all other possible outcomes. If the outcome varies from the anticipated, there are several possibilities to consider. 

No Band:If no band is visible after staining the SDS-PAGE gel, it could suggest that the spike protein has not been expressed at all, possibly due to unsuccessful agro-infiltration or an issue with the protein extraction process.

Multiple Bands: If multiple bands appear, it could suggest that the protein is being expressed, but that there may be post-translational modifications, such as glycosylation or phosphorylation, leading to multiple forms of the protein. It could also indicate that the protein is breaking down into smaller fragments, or it might suggest contamination with other proteins.

Single Band at the Wrong Molecular Weight: If a single band is visible, but it doesn't match the expected molecular weight for the spike protein, it could suggest that the protein is not being processed correctly after synthesis, leading to a larger or smaller than expected protein. Alternatively, it could mean that a different protein has been expressed, either due to an error in the agro-infiltration process or due to an issue with the plasmid used.

Remember, these variations in outcomes are part of the scientific process. They allow us to refine our methods and hypotheses, and sometimes they may even lead us to new discoveries.

6. What is the ALTERNATIVE hypothesis? In another words, if the experiment results don't match expectations, what do other POSSIBLE results may imply?

The Alternative hypothesis is also a statement sentence, not a open-end question sentence.

7. What are the key findings from the experiment(s)? Interpret these yourself, don't just echo the authors.

This is the pivotal phase in the entire procedure. Approach it with the meticulousness of a detective. Avoid jumping to any conclusions immediately. Instead, begin with minor details, accumulating ALL THE TINY, UNBIASED FACTS that you've noticed, like a CSI (Criminal Scene Investigation) agent. For example, in Fig. 2 of Paper 1, collect evidences like "there is a band at ...kDa position", "The intensity of the ...kDa band is heavier than the one at ...kDa". "in Western blot figure, there is a band observed at ... location", etc. Forget about any conclusions that you attempt to rush at, wait until you have ALL the evidences/findings. 

8. What's your conclusion based on your answers to question 7?

Now considering all the findings collected, please draw a conclusion. You may have one conclusion or multiple conclusions. It would be fascinating if you could have a single conclusion that are supported by ALL the evidences that you have collected.

9. Does your conclusion match the authors'?

In the manuscript, hunt for the sentences that reference the figure number. For instance, if you're examining Figure 2 in Paper I, search for the sentences that include "(Fig. 2)" or "(Figure 2)". These are typically tied to the data represented in that specific figure. This strategy will likely lead you to the authors' descriptions of their discoveries and their corresponding conclusions.

10. If you were in the authors' shoes, would you design the same assays?

To tell you the truth, I like this part the most - it's a real intellectual challenge. You see, there isn't just one method to test a hypothesis, there are always several. When scientists draw up their grant proposals, they often propose a variety of ways to put the same hypothesis to the test, being it via genetic approach, or biochemical approach, or cellular biology approach, etc. Take this as an example: if we want to investigate whether the mutant S protein physically similarly as its wild-type counterpart interacts with ACE2 protein under specific conditions, we could suggest a range of strategies to explore their interaction both inside a living organism (in vivo) and in a lab setting (in vitro). This could include techniques like the Yeast two-hybrid assay, Pull-down assay, or even the ChIP-MassSpec method, among others. Further, they would propose to test the biological importance from other perspectives as well, such as from a genetic perspective, a biochemical, a biophysical or a physiological one, etc.

11. How could the conclusion obtained feed into the big picture or the overall theme of the paper and the research field?

Reflect on the conclusions you've reached and those given by the authors. How might they, together, augment the current model? Do these insights reconcile the previously discussed observations that didn't seem to fit? If these new conclusions are woven into the current model, what would the resulting model look like? Could this new model provide a basis for predictions? How might it influence the research field?

When you first started reading the research article, I advised you to initially skip the abstract. Now, it's time to delve into it. You'll likely notice a somewhat fixed hourglass structure in the Abstract. The opening sentence typically sets the scene with a broader context, which then gets narrowed down to the specific research question in the following sentences. Phrases like "In this study, we ...." or "We tested ...." often indicate the beginning of the section where research methods and conclusions are outlined. In the very last sentence of the Abstract, it typically rounds off with the authors' interpretation of how their findings could fit into the larger research landscape.