Studying and working on something specific, it is hard to find books to read in order to understand them thoroughly. Especially, when doing the new experiment by using old knowledge, it is impossible to get new results. In scientific aspect, the way to find new knowledge is by reading updated research articles, which scientists can improve their in depth knowledge and complete their specific experiments. But how many scientist wants to spend their time to study other field’s research articles? We think the answer is only a few. If we regard this as a problem, one of the ways to solve it is by having this class.
Each lecturer gave us an interesting article in their field, whereby most of these articles are the ones we weren’t familiar with. So our work was reading and understanding the articles. Because of we have the chance to read new topic, this class made us change our attitude on choosing only thesis related articles to read. So what we have learned from Current Topic? Let’s see in next sections.
RNA-methylation-dependent RNA processing controls the speed of circadian clock
Lecturer : Asst. Prof. Dr. Sarin Chimnaronk Date : 11 October 2017
Circadian clock is a biochemical process in many organisms that relates to solar time. Normally, the light gets through organisms by their eyes. The light signal can go to hypothalamus which controls circadian period in organisms’ body and controls metabolic gene expression. There are many rhythmic genes such as Per1, Per2, and ARNT1. When they focus on the rhythmic genes, the researchers found that 10% of liver transcriptomes are driven by circadian clock. There is only a fifth which is driven by De novo transcription. That means four out of five genes are driven by mRNA modification. For this reason, it led us to the question: is mRNA methylation can control circadian clock period?
The methylation is a process where methyl are transferred from methyl donor to other molecules. For example, S-adenosylmethionine (SAM) gives methyl group to other molecules then turns into S-adenosylhomocysteine (SAH). The methylation potential is a ratio between SAM/SAH. When methylation potential decreases, inhibition of methylation can occur. From this article, methylation was inhibited by 3-deazaadenosine (DAA) which can prevent SAH hydrolysis. This inhibition lead to decreasing of methylation potential and then methylation inhibition. After treating peripheral cells and central nervous cells with DAA, it results in inhibition of methylation which can elongate circadian period significantly.
The investigation was done by adding methyl at the m6A site, which is a methylation site abundant in mRNA. The results showed that DAA can also inhibit methylation at the m6A sites in mRNA. To clarify that methylation was the actual point which can elongate circadian clock, gene knockdown and overexpression were performed. The METTL3 gene which played role in methylation at the m6A sites was knocked down as a result in period elongation. On the other hand, the period was shortened when this gene was overexpressed. To compare among cytoplasm mRNA, nuclear mRNA and Pre-mRNA contents in the same period, they found that inhibition of methylation at m6A sites delays mRNA export from nucleus to cytoplasm. In conclusion, the methylation can elongate circadian period but how exactly does this process can elongate the period is still unclear in this article.
Ethics in Biological Research
Lecturer : Asst. Prof. Dr. Apinunt Udomkit Date : 18 October 2017
We heard about ethics for a long time since we were kids and more often in Social and Humanity subjects. However, do we know the real definition of this word? Somebody says no, somebody say yes. Let's see what we got from this lecture.
The lecture began which the definition of ethics “well-founded standards of right and wrong that prescribe what human ought to do'' (Velasquez M, et al.). It is very important for the being a scientist and not to make scientific research misconduct. We learned that ethics is related with our research i.e. planning, doing experiments and reporting the research. All these steps must be done with prime concerns about the ethics. The example of misconduct is editing the pictures or changing the data when the results are not the same with our expectation.
One of the most important parts about ethics in our research misconduct is plagiarism. Plagiarism is the action or practice of taking someone else's word and ideas and pasting it off as one's own. To be a good scientist and to prevent declining from publishing the paper, we must avoid the plagiarism. Then, we learned how to avoid plagiarism and practiced how to report the data in order to be accepted.
These are all of what we learned in this lecture. Although, we got new knowledge about the ethics that affects our research, it would not work if we only learned and forgot it. Just always keep in mind to be honest and responsible.
Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen
Lecturer : Nitwara Wikan, Ph.D. Date : 25 October 2017
A large outbreak of Zika virus (ZIKV) occurs in 2014 and it makes a major concern to the World Health Organization (WHO). The disease associated with this virus called Guillain-Barré syndrome and causes neural disease. However, there is currently no drug approved to prevent ZIKV infection. Therefore, finding the compound that can prevent the effect of ZIKV can be achieved by developing a new drug that can improve the quality of therapy.
From the previous study, they found that when human neural progenitor cells (hNPCs) was infected by ZIKV, caspase-3 activity is increased and it causes the cell death. Thereby, in this project, they identified neuroprotective (inhibitor of caspase-3 activity in ZIKV infected cells) and antiviral compound. They started with developing the high-throughput compound-screening approaches, by comparing between caspase-3 activity approach and cell viability assay. Measuring caspase-3 activity is a better assay than cell-viability assay. Then, they selected the primary hit compound that suppressed the ZIKV-induce caspase-3 activity. Emericasan is a caspase inhibitor compound that was chosen because it is the most effective anti-cell-death compound. After they treated Emericasan in ZIKV-infected human astrocytes and hNPCs cell, they measure caspase activity and normalized cell viability. The results show that the more concentration of Emericasan (the data show the highest concentration is 10-4 M), the lower the caspase activity and the more cell viability. From these results, they claimed that Emericasan is an effective neuroprotective compound. The other two compounds selected from primary hit lists that can inhibit ZIKV infection are niclosamide, which is the drug for curing infection from worms, and PHA-690509, which can inhibit cyclin-dependent kinase inhibitor (CDKi). Both compounds can inhibit replication of infected cell. They used western blot to analyse the expression of ZIKV protein (NS1) attached with anti-ZIKV antibody. The results showed that the amount of NS1 decreases when increasing the concentration of both niclosamide and PHA-690509. In contrast, in Emericasan treated infected cells, it showed no decrease of NS1 protein, which means that Emericasan can only be neuroprotective compound. Niclosamide can neutralize endolysosomal pH and relates to change in pH in membrane fusion which is the an important step in common virus entry pathway. For CDKi, they assumed that some CDKs in host cell have important roles in viral replication. When the CDK is inhibited, it inhibited replication. Finally, they treated infected cell with both compounds, i.e. niclosamide and PHA-690509. However, the results were not clear and this experiment is still ongoing.
From this paper, it makes me realized how serious this virus is and it inspired me with the techniques of how to find new drug and hope we can apply this technique to other disease.
Quantitative imaging of gut microbiota spatial organization
Lecturer : Dr. Poochit Nonejuie Date : 1 November 2017
The gastrointestinal tract provides ecosystems for a variety of microbes. The diverse organisms have organized their location that will be proper to the host tissue. Therefore, mislocation can lead to abnormality to the host gut. In colon, inner mucus layer can prevent bacteria from directly contact epithelium and outer mucus layer is for providing nutrients for microbes. From this knowledge, lacking of microbiota-accessible carbohydrate (MACs) which microbes use for metabolism, can affect intestinal microbiota localization. To quantitate that, confocal microscope was used in combination with developed image analysis software, BacSpace.
To perform the experiments, standard MACs and MACs deficient (MD) diet were treated to germ-free mice. After treating, the mice were colonized with (1) Bacteroides thetaiotaomicron, a mucus utilizing microbe, (2) human microbiota and (3) B. thetaiotaomicron with Salmonella typhimurium to observe bacterial distribution. The mice were sacrificed and gut tissue were collected and then observed.
In colonize B. thetaiotaomicron, there is thinner inner mucus layer in MD diet compared with standard diet which led the bacteria to get closer to the host epithelium with high density. Not only the B. thetaiotaomicron colonized bacteria can be affected by the lack of MACs in term of mucus thickness and microbe localization, but also the human gut microbes and in S. typhimurium which consumes sialic acid from B. thetaiotaomicron. However, there were no proximity-induced in these 2 organisms. Furthermore, to study how the mucus affects microbial localization in bacterial communities, mice gut which were treated with standard diet illustrated that bacteroidales and firmicutes clusters excluded each other and the bacteroidales resided closer to host epithelium than firmicutes. After changing to MD diet in the same group of mice for 10 days, the 2 orders of microbes seems mixing to each other. In inflammatory observation by detecting antimicrobial lectin regenerating islet-derived protein 3 beta (REG3β) which refer to inflammation, there were REG3β enrichment in MD diet mice more than standard diet mice. From these results, localization of microbes can refer to host’s mucus status and the localization also can lead to changing of REG3β expression in intestine.
When the environment of microbiota in gut had some change in biochemical component, mucus or host tissue can lead to microbiota organization. Lacking of MACs lead to thinning of the mucus layer, that might lead the bacteria to get closer to the host epithelium. Then, an increasing of antimicrobial protein REG3β expression occurs, which can indicate inflammation in gut.
Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols
Lecturer : Dr. Chalongrat Noree Date : 8 November 2017
In this class, we had learned about compartmentalization and played the quiz game about this paper. Compartmentalization means the formation of cellular compartments which contains the boundary. This research published by Avalos JL, et al. (2013) took an advantage of this characteristic of cell compartmnetalization in order to develop the production of branched-chain alcohols in yeast.
Mitochondria contains intermediates of many metabolic pathways and has an optimal environment for the maximal activity of many enzymes. Due to the small volume of mitochondria, they could concentrate substrates and restrict intermediates leading to faster rate of a reaction and more productivity. This research took the advantage of characters of the mitochondria environment for biofuels production in yeast (Saccharomyces cerevisiae), including isobutanol, isopentanol and 2-methyl-1-butanol. The upstream part of isobutanol pathway, valine Ehrlich degradation pathway, is located in the mitochondria, whereas the downstream part is located in the cytoplasm. Therefore, the problem was the difficulty of the transportation of all enzymes across the membranes because it could reduce productivity and intermediates might be lost to competing pathways. To avoid this problem, this researcher developed pJLA vectors for overexpression of all enzymes in this pathway which were targeted to mitochondria. The pJLA vectors were constructed with different promoters, including TDH3, PGK1 and TEF1 promoters, and the N-terminal mitochondrial localization signal from subunit IV of the yeast cytochrome c oxidase were targeted to the downstream enzymes. Then, the production of isobutanol, isopentanol and 2-methyl-1-butanol was measured for comparison between the enzymes targeted to the mitochondria and the cytoplasm. The results showed that the production of isobutanol increased by 260% due to downstream enzymes targeted to mitochondria (α-ketoacid decarboxylase, α-KDCs, and alcohol dehydrogenase, ADHs), compared to the enzymes targeted to cytoplasm with 10% improvement. Like isobutanol production, the overexpression of α-KDCs and ADH increased the production of isopentanol and 2-methyl-1-butanol when they were targeted to mitochondria. This research has showed the benefits from mitochondria compartmentalization into mitochondria in Ehrich pathway. Thus, the compartmentalization has a potential application in improving availability of enzyme or intermediate from other metabolic pathways.
The human cancer cell active toxin Cry41Aa from Bacillus thuringiensis acts like its insecticidal counterparts
Lecturer : Assoc. Prof. Dr. Panadda Boonserm Date : 15 November 2017
In this class, we learned how to make a note for reading paper and present the results and significance of a paper. The study published by Krishnan V, et al. (2017) focused on Cry41Aa (parasporin 3), a subset of Cry toxins that has showed the activity against human cell lines. The aims of this paper were to investigate the activity of Cry41A on HepG2 cells and to determine the specificity of this toxin by the ricin domain. The results showed that activation of Cry41Aa by trypsin could reduce viability of HepG2 cells by almost 50%, whereas the ricin-deleted Cry41Aa had no significant difference to the activated Cry41Aa. It was indicated that the ricin domain did not determine the specificity of the toxin. The full-length Cry41A could significantly affect cell viability within the first few hours after treating HepG2 cells with Cry41Aa. Cry41Aa induced a rapid reduction in ATP levels and an increasing of H2O2 in HepG2 cells, which suggested that the effect of Cry41Aa on HepG2 cells were mediated by pore-forming capability via necrosis pathway. The membrane damage was assessed after treating HepG2 cells with Cry41Aa. The results showed that a significant effect was seen 90 min after treatment and reached to the level seen with digitonin-based lysis solution after 5 h. Moreover, the result showed the increasing of the breakdown of the cell membrane at the similar level as the Triton X-100 did in 5 h. The result from microscopic analysis showed that HepG2 cells were swelling after treated with Cry41Aa for an hour. For the investigation of aromatic amino acids in loop 3 of domain II of Cry41Aa, the amino acid substitutions, including F509Y and W511F, would retain enzymatic activity which supported that aromatic amino acids in this region are important for toxicity of Cry41Aa.
In conclusion, the author of this paper found that the deletion of the ricin domain did not significantly affect the activity of the toxin against the HepG2 cells. So this region did not have function with the expression of toxin. Moreover, Cry41Aa kills cells by making pore on the cell membrane via necrosis pathway. This toxin decreased the metabolic activity, made cell swelling and causes cell membrane damage. The exposed loop region might be involved in receptor binding and hence it is important for the activity of Cry41Aa against HepG2 cells.
What we have learned from this course were summarized into this collaborative writing class instructed by Ajarn Duangrudee. In the first class, she taught us the new tools for collaboration. It is very useful for group work and it is a convenient tool to improve the quality of our writing with many function of program including grammar checking. We liked it so much. For Collaborative writing class, it helps us to practice reading comprehension in English, practice writing and using English by helping one another to do work in group. As we know, English is an important language in the world. Working in team helps us to adapt ourselves to other people. Each lecturers gave us some tricks to read article so much faster, clearer and more understandable than before.
In summary, this course inspired us with new knowledge from different fields. It makes us realize what is going on in the world and reminds ourselves to keep up to date. The new discoveries are coming daily and makes us get one new hobby, which is reading scientific literature.
1. Fustin J-M, Doi M, Yamaguchi Y, Hida H, Nishimura S, Yoshida M, et al. RNA-Methylation-Dependent RNA Processing Controls the Speed of the Circadian Clock. Cell.155(4):793-806.
2. Velasquez M., Andre C., Shanks T., S.J., and Meyer M.J. A definition of ethics in terms of standards such as rights and fairness. California: Santa Clara University; 2010 [cited 2017 Oct 18]. Available from: https://www.scu.edu/ethics/ethics-resources/ethical-decision-making/what-is-ethics/.
3. Xu M, Lee EM, Wen Z, Cheng Y, Huang WK, Qian X, et al. Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen. Nature medicine. 2016;22(10):1101-7.
4. Earle Kristen A, Billings G, Sigal M, Lichtman Joshua S, Hansson Gunnar C, Elias Joshua E, et al. Quantitative Imaging of Gut Microbiota Spatial Organization. Cell Host & Microbe.18(4):478-88.
5. Avalos JL, Fink GR, Stephanopoulos G. Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols. Nature biotechnology. 2013;31(4):335-41.
6. Krishnan V, Domanska B, Elhigazi A, Afolabi F, West MJ, Crickmore N. The human cancer cell active toxin Cry41Aa from Bacillus thuringiensis acts like its insecticidal counterparts. The Biochemical journal. 2017;474(10):1591-602.