On page 209 in the article, the authors discuss the genetic analyses used to study fat modulation. I am confused about the screen in animals "that are deficient in fat metabolism as a result of loss of fatty acyl-CoA synthetase genes." These animals were susceptible to fat reduction by some reducing compounds. What does this really show? Wouldn't they already have reduced fat because they are deficient in fatty acyl-CoA synthetase genes? Or do I have something backwards here?
Also, this paper is one of many that we have read indicating a possible genetic link to obesity. How do you think these findings will affect the public and the health care and insurance industries in the future?
We talked briefly last week about the lack of specificity in Nile Red staining. I wonder, in this instance, if the authors have successfully ruled out the possibility that the compounds they identified (especially those that do not have a phenotype in other organisms) are simply observed because of degradation or more efficient Nile Red staining? In other words, have the authors successfully shown that the compounds they identified interact with pathways important for energy homeostasis, or do you think it's possible that they are merely interacting with Nile Red in some significant way?
In the discussion section of the Lemieux, et al. paper, the authors note that their results "stand in contrast to several recent publications that have implied that vital dye staining is nonpredictive of changes in global fat metabolism in C. elegans." Were these other papers claiming that Nile Red has limitations as a marker for fat metabolism as we discussed a little last week?
The authors justified their results over these other papers by pointing out that dye-based imaging was more sensitive than triglyceride extractions. What do they mean by this?
In the RNAi paper last week, we discussed that C. elegans can take up RNAi by being soaked in an RNAi-containing solution. Does the use of small organic molecules in C. elegans use a similar methodology? If so, how would this work with larger organisms? Also, in larger organisms, would the organic molecules need to be targeted to a certain location where the protein of interest was most prominent?
Last week we learned that RNAi is a fairly effective technique for silencing the expression of a particular gene. How comparable is chemical genetics to RNAi in terms of efficacy? Does it generally prevent protein function as well as RNAi, or does that depend on the particular protein/chemical?
In the review paper, the author, especially in the first few sections, seems generally disappointed with the current chemical genetics methods. Even the ones that he chooses as his 'favorites' still have drawbacks. This begs the questions, how reliable or useful are these methods? And is it a feasible possibility to accomplish all of the advancements/changes that the authors proposes as necessary for more detailed and intricate results?
In the paper they had mentioned they did an RNAi knockdown of genes predicted to encode kinases and transcription factors. It had stated that only 70% of these predicted genes were tested with RNAi. Was this because the RNAi library did not have the other 30% of the genes or is there some other reason why they didn't test all of the predicted genes.
In the research paper by Lemieux et. al., they were able to show that several small molecule compounds, that utilized specific signaling pathways, showed a decrease in fat storage in C. elegans (ex. F21) as well as in mice and fly tissue, and an increase in lipid droplets of other specific small molecule compounds (ex. A15). By seeing the same correlation among other organisms in relation to C. elegans, is it possible that these compounds will function in the same way by increasing and decreasing fat storage in other model organisms such as zebra fish, or even humans? The article also mentioned that F17, which decreases fat storage, affects the AMPK pathway in other organisms besides C. elegans, but never broke down what the AMPK pathway is. So, what exactly makes the AMPK pathway important to this study, and how does it function?
After reading the article and the review I am having a hard time understanding what the method of chemical genetics actually entails. My understanding is that it uses libraries of data to compare proteins in a molecule with mutations to the wild type, however I am not sure if this is accurate. What is the actual process of chemical genetics?
In the primary article, they mentioned that there isn't high correlation between feeding behavior and fat storage levels after noting that there were some C. elegans with decreased fat storage and increased feeding behaviors. They list one reason for why this might be but could you think of another reason? Could it have something to do with the small molecules?
The review paper talks about how a molecule library should be structured; whether that be large with plenty of molecules or small with a few related molecules that are more likely to be used for phenotypic studies. Do you support the idea that chemical libraries should only be limited to molecules that we already know will cause certain phenotypes on cell components?
In the review paper “Exploring biology with small organic molecules”, the author explains using small molecules to understand biological systems further. He gives two examples of chemical libraries; “focused libraries” and “diversity- oriented libraries”. However, even though the author explains chemical library pretty well, i am still confused the process of chemical genetics. And how chemical libraries can specifically be used.
We have learned that more than just regulation of homeostasis can play a role in fat storage/usage (neurotransmission, genes like tubby, etc.). How can we compensate for the difference in these factors between nematodes and mammals in research? Entire animal screening is a solid approach for addressing how a molecule regulates fat storage in vivo rather than in vitro, where not all of the factors regulating an organism's body are in play, but what must be done in order to comparably study something as complex as fat regulation in organisms that are so different?
On page 207 of the article, "A whole-organism screen identifies new regulators of fat storage" by Lemieux, et al., the authors discuss the hit rate of their in vivo screen. They stated that, "the hit rate of this screen is high relative to in vitro high-throughput screens..." Why is it that an in vivo screen would yield a higher hit rate than one in vitro?
Was the phenotypic screening of chemical libraries in this paper forward or reverse genetics? I don't quite get the flow of identification of drug targets. Since chemical genetics deal with signal transduction, do you think it has a high potential for discovering new cancer treatments?
The authors mention that C. elegans is a good model because "core mechanisms are largely conserved". Is it okay to generalize like this when studying mechanisms and pathways? Could this statement potentially hinder future studies because of the assumptions that are being made?
Do you think that this article could have used more abbreviations?
Is the basic plan here generalizable to determining the function of other genes where the end of the pathway in question is not stainable by nile red? It seems like doing the small molecule screen is straightforward, but only useful if there is a visible phenotype change at the end
The paper by Lemieux et al. states that the fat reduction phenotype induced by F17 is partially dependent on the function of a transcription factor called K08F8.2. Is anything known about the role of this transcription factor in fat regulation? Is there a mammalian homologue? What does this transcription factor bind to? Does knocking out (or RNAi) of this transcription factor lead to increased fat in either C. elegans or other organisms?