What we have learned from

Current Topics in Molecular Biology classes (MBMG 601) ?

  1. MR. Todsapol Kornsri 5936117 MBMG/M
  1. Contents
  1. Dr. Poochit’s class
  2. Assoc. Prof. Panadda’s class
  1. Organization
  2. Conclusion and Discussion
  1. Miss Wipaporn Khanom 5936118 MBMG/M
  1. Contents
  1. Assoc. Prof. Apinunt’s class
  2. Dr. Nitwara’s class
  1. Conclusion and Discussion
  2. Grammar and Spelling
  1. Miss Atitaya Hitakarun 5936424 MBMG/D
  1. Introduction
  2. Contents
  1. Asst. Prof. Sarin’s class
  2. Dr. Chalongrat’s class
  1. Organization
  2. Mind mapping

Mind mapping about the responsibility

Introduction

For the first time when we have heard that we have to study Current Topics in Molecular Biology course, what do we think and imagine about this course? From our discussion, we think we will have a chance to learn about the reading techniques from various scientific papers and those high impact factor papers such as Nature, Cell, Lancet  must be very complicated.  It is quite a difficult course for us due to our experiences.

For the course orientation, we were divided into 2 groups, which consists of 3 persons, and received one assignment about collaborative writing from the knowledge that we learned from this course. Ajarn Duangrudee Tanramluk, the course coordinator, suggested us about the using of Google tools for collaboration. This course aims to provide that students with analytical skills to understand scientific literature based on molecular biology principles and theories. The course schedule consists of 8 lectures that is composed of different research fields, teaching methods, and assignments. With these reasons, we attempted to understand the details of each article. First of all, we read individually and reviewed the knowledge background of each article, then followed by group discussion before the class.

                After finishing this course, we hope that it will help us improve the writing skills especially when we try to explain some difficult knowledge to the others who either have or do not have molecular biology background.

Contents

1st class: October 11th, 2017

Topic: RNA-Methylation-Dependent RNA Processing Controls the Speed of the Circadian Clock (1)

Teaching staff: Asst. Prof. Sarin Chimnarong

                This is the first class of this course. We received an article about “RNA-methylation-dependent RNA processing controls the speed of the circadian clock” that is a hot issue as this time. This article is a high impact factor paper with journal impact factor (JIF) 30.410 that is very complicated for students’  individual reading.  Ajarn gave us this paper on the next day after the Nobel prize in physiology or medicine was awarded to 3 American scientists for discovering the circadian rhythm which controls the biological clock. It was quite a surprise for us because the teacher can explain about it very clearly.

                In the first hour of the class, Ajarn taught us about ‘How to read paper?’ which is interesting and very helpful for us. He advised us to focus on the abstract of the paper and summarized the article in mind mapping.

                Next, Ajarn briefly but clearly explained and concluded each part of the paper which are the introduction, the hypothesis and the main results in each figure for  us.  

                Finally, we summarized and shared ideas that we learned from this week. Everything in this paper is new, and the knowledge we obtained might led us to solve difficult problems in different disease, etc.  

2nd class: October 18th, 2017

Topic: Ethics in Biological Research

Teaching staff: Assoc. Prof. Apinunt Udomkit

This class, we have learned about the research ethics which is very important for the researchers. Ethics is จริยธรรม in Thai. Velasquez et al. said that "Ethics consists of the standards of behavior our society accepts" and "Ethics refers to well-founded standards of right and wrong that prescribe what humans ought to do, usually in terms of right, obligation, benefits to society, fairness, or specific virtues." While the moral (or คุณธรรม in Thai) is the principles on which one's judgments of right and wrong are based.

At the first time we start doing our research, we have to review the previous studies, and then grab the concepts of the study to answer the hypothesis. When we finish the thesis, we have to compare and discuss our results with others. All of these steps of research, we must have ethics. Scientific research misconducts including fabrication which is presenting the data without doing any experiments, i.e. makeup data, falsification which is presenting some parts of data to convince other people, and plagiarism which is taking other people’s ideas and words to present as it is your own. The examples of ethical misconducts are divided into 2 main parts in this class, which are image manipulation and plagiarism. Image manipulation is image adjustment such as enhancing the brightness or contrast, removing the background and cropping. In molecular biology field, most of the data will be published or presented in the image such as gel bands. The images of the gel can be manipulated but we must adjust the whole image. The background can be reduced but every band on the gel should remain in the image. When we combine the images together, we have to make it clear that these images are from different experiments by putting the separation lines. One of the most important parts is plagiarism, which is to take another's works, words or ideas as your own without giving any credit or acknowledgment. To avoid plagiarism, we can provide citation or quotation, paraphrasing, and summarization.

3rd class: October 25th, 2017

Topic: Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen (2)

Teaching staff: Dr. Nitwara Wikan

Zika virus (ZIKV) is a flavivirus that can be transmitted to human by the bite of Aedes species mosquitoes. The recent outbreak has begun since 2013 which the World Health Organization (WHO) declared as a global health concern. The most important problem of this ZIKV infection is that the infected pregnant women can pass on the virus to her fetus who may develop microcephaly which mean her baby will have abnormally small head size. Guillain-Barre syndrome may occur in the infected adults and may cause paralysis from the destroyed myelin sheath.

In this paper, the researcher has screened about 6000 drugs which comprised of both approved drugs and candidate compounds that pose the efficiency to inhibit ZIKV infection and suppress active caspase-3 activity which are induced by the viral infection. Moreover, they quantified the two methodologies which are an ATP-content assay for cell viability measurement and the caspase-3 assay for  detection of ZIKV-induced caspase-3 activity. The results showed that caspase-3 activity measurement is a better method for high-throughput screening compounds than the cell-viability assay.

They used ZIKV strain MR766 (Uganda strain) to infect with human neural progenitor cells (hNPCs) and glioblastoma cell lines or SNB-19 cells. Next, they screened the collected compounds. In this step, they could identify the compounds which can decrease the caspase activation and apoptosis induced by ZIKV by either preventing the cell death or suppressing viral replication. They founded that emricasan, which is the pan-caspase inhibitor in phase 2 trials to reduce the hepatic injury and liver fibrosis from chronic HCV infection, shows neuroprotective activity for hNPCs but can not suppress viral replication. For niclosamide and PHA-690509, they reported that these 2 drugs have a possibility to inhibit ZIKV infection at a post-entry step of viral replication cycle. Niclosamide, an FDA-approved drug to treat worm infection, showed IC50 (half-maximal concentration) at 0.37 uM whereas PHA-690509, which is a cyclin-dependent kinase inhibitor (CDKi) compound, showed IC50  at 1.72 uM. Moreover, they also combined two compounds together which emricasan showed the neuroprotective activity and the PHA-690509 showed the anti-viral activity. The results showed that the combination of these two compounds provided the additive effect to inhibit caspase-3 activity in SNB-19 cells. Furthermore, in this publication, the researchers discussed about the effect of this drug on pregnant women. They said that CDKi (PHA-690509) and its derivatives are very beneficial for the non-pregnant women but it can not be used to decrease the risk of Guillain-Barre syndrome in pregnant women. To prevent this neurological complication, niclosamide is suggested.

4rd class: November  1st, 2017

Topic: Quantitative Imaging of Gut Microbiota Spatial Organization (3)

Teaching staff: Dr. Poochit Nonejuie

The human gastrointestinal (GI) tract represents one of the largest interfaces between the host, environmental factors and antigens in the human body. Our gut contains tens of trillions of beneficial microbials, including at least 1000 different species of known bacteria with more than 3 million genes. Microbiota has many advantages such as it helps the body to digest certain food that the stomach and small intestine cannot to digest. It plays an important role in the immune system by performing a barrier effect. Thus, gut microbiota plays an important role in host health maintenance and disease pathogenesis. In this paper, they focus on developing some software to study the composition and the diversity of host-associated microbial populations in gastrointestinal tract. Within the lumen, microbes can cling to one another, partition within or be excluded from difficult to detect biochemical gradient or attach to scaffolds of food particles, mucus, or sloughed epithelial cells.  This research utilized a combined mucus preservation and labeling methods with confocal microscopy to generate image by using mouse as a model to study microbiota accessible carbohydrates from the diet. After that, they developed computational tools that can automate stitching, subtraction of non-specific signal and localization measurements in the gastrointestinal environment. Then, their finding lead to the development of BacSpace software to help mitigate error that are introduced through experimental manipulation. The results show that, in thinner mucus and the fiber protective mucus layer, there was an increase in proximity of microbes to epithelium and a heightened expression of antimicrobial innate protein REG3b inflammatory markers. In addition, they performed cell to cell measurements in defined and complex communities. The MAC-rich standard diet is the intestinal microbiota containing large clusters of related microorganisms. The researchers determined  that by identification of particular species within the cluster by using more specific labeling strategies or by creating a defined communities in gnotobiotic mice, they can elucidate the factors controlling this microbiota organization. Thus, microscopy and image analysis are underutilized tools in the study of the gut microbiota. With an improved histological methods and computational tools provided here, this method will be critical for defining the significant and biologically meaningful spatial relationship within biopsies from healthy and diseased human gut tissue.

 

 

5rd class: November 8nd, 2017

Topic: Compartmentalization of metabolic pathway in yeast mitochondria improves the production of branched-chain alcohols (4)

Teaching staff: Dr. Chalongrat Noree

         Saccharomyces cerevisiae is a common yeast used to molecular genetics  and metabolic engineering to produce the compounds i.e. isobutanol, isopentanol, and 2-methyl-1-butanol, which are alternative sources for the transportation fuels production. This article focuses on the main product, the isobutanol compound, which is synthesized via the upstream and downstream of isobutanol pathways. Normally, the isobutanol biosynthesis is divided into two parts, the upstream part (from pyruvate to α-ketoisovalerate) in mitochondria and the downstream part (from α-ketoisovalerate to isobutanol or Ehrlich pathway) in the cytoplasm of yeast cells. The hypothesis of this study is based on the assumption that if the researcher can relocate the complete pathway, especially α-KDC and ADH, into mitochondria or can reduce the loss of intermediates to competing pathway, the production of these biofuels, isobutanol, isopentanol, and 2-methyl-1-butanol, would be increased. To test this hypothesis, the investigators have constructed a set of vectors, pJLA vector series, which carried the partial or complete genes of metabolic enzymes involved in isobutanol pathway. After that, these plasmids were cloned and transformed into yeast cells. Then, they observed the viability of colony size, growth rate, and measured the fuel alcohols production in both high and low-cell-density fermentation. In addition, they also measured the total protein in each subcellular fraction and detected those proteins with specific antibody. The investigators found that the overexpression of isobutanol pathway completely targeted into mitochondria resulted in higher titer of isobutanol production, whereas the overexpression of the downstream pathway in the cytoplasm exhibited much lower titer of product, which was similar to the results of overexpression of the upstream genes in mitochondria. For the isopentanol and 2-methyl-1-butanol, the production of these alcohols presented similar trends to the isobutanol production that the higher titer came from the overexpression of both α-KDCs and ADHs in mitochondria. Moreover, the relocation of the downstream enzymes to mitochondria that led to the increasing amount of biofuels, was a result of the increasing concentration of local enzymes in mitochondria that lead to the other benefits. This study provides an alternative way to improve the production of biofuels since this technique uses a smaller amount of organisms to obtain more products, especially the production of isobutanol that showed the highest titer compared with the previous reports. The knowledge about metabolic engineering could be applied to use with other organisms such as plants and cyanobacteria.

6rd class: November 15nd, 2017

Topic: The human cancer cell active toxin Cry41Aa from Bacillus thuringienesis act like its insecticidal counterparts (5)

Teaching staff: Assoc. Prof. Panadda Boonserm

 

Bacillus thuringiensis is the key insecticidal bacterium found in environment that can be used to control various insects and nematodes. It can produce toxin that can target at the epithelial cells of insect gut and lead to death of the organism. However, the new alternative toxin for killing insect is still an important topics to study in order to find new toxin that can used the same mechanism but does not affect human. One alternative is a Cry41Aa that are found within a three-gene operon, which are ORF2 that encodes the functional portion of the toxin, ORF3 that encodes a protein believed to be important for expression within Bt and ORF1 that has no known function. In this paper, the researchers aim to study activities of the Cry41Aa toxin with or without ricin domain. The ricin domain play a specificity role on HepG2 cell throughout the toxicity mechanism. To prove this hypothesis, firstly, they cloned cry41Aa with and without ricin domain by designing two PRC primers to amplify ORF2 and ORF3 from the cry41Aa operon and incorporated suitable restriction site. Then, they studied toxin expression and purification of cry41Aa by gel filtration column. The fractions were analyzed by 7.5% SDS and the protein concentration was determined by the Bradford method. After that, they tested the toxicity with HepG2 and HeLa by using CellTiter blue assay. Next, they induced the apoptosis pathway in HepG2 cells by using Caspase-Glo 3/7 assay. Then, they tested the membrane integrity in HepG2 cells by using Cell Tox Green and tested H2O2 production (ROS level) in HepG2 cells by using ROS-Glo H2O2. Finally, they performed single amino acid substitution mutants with cell viability in HepG2 by using CellTiter blue assay. The results showed that the deletion of ricin domain did not significantly affect the activity of the toxin against human cell line and Bt Cry41Aa did not induce apoptosis but it acts as a pore-forming toxin. Therefore, the structural part of loop 3 in domain II is an important for studying the nature of interaction between the toxin and the host cell receptor.  They can predict the specificity of toxin from the amino acid sequence and can lead to a better understanding of the mechanism of Bt toxins target to a particular host.

References

  1.  Fustin, J. M., et al. (2013). RNA-methylation-dependent RNA processing controls the speed of the circadian clock. Cell 155(4): 793-806.
  2.  Xu, M., et al. (2016). Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen. Nat Med 22(10): 1101-1107.
  3.  Earle, K. A., et al. (2015). Quantitative Imaging of Gut Microbiota Spatial Organization. Cell Host Microbe 18(4): 478-488.
  4.  Avalos, J. L., et al.(2013). Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols. Nat Biotechnol 31(4), 335-341.
  5.  Krishnan, V., et al. (2017). The human cancer cell active toxin Cry41Aa from Bacillus thuringiensis acts like its insecticidal counterparts. Biochem J 474(10): 1591-1602.