Techniques and Application in 21st Century Problems from Current Topics in Molecular Biology Classes  

1. Introduction

As a researcher, we can not live without scientific paper which have been updating every second. When we are searching for a paper, the interesting parts are in methods or techniques that were used. Molecular techniques have been developed very fast to become more efficient, less time-consuming and easier for utilization. In this year (2018) during the Current Topic class, we had learnt many interesting topics, which included molecular techniques and applications to figure out the research question. One of the most important things as a researcher and a student is that we should concern about plagiarism in our work and also other’s work. Some publications might show artificial results so we have to be careful and cautious when we read the paper. This report will show you about principles of molecular techniques and example of application of using those techniques.

2. Contents

2.1. Aj. Sarin’s class: Whole Exome Sequencing

Whole exome sequencing (WES) is one of the techniques for sequencing of the exome or all of  coding genes in the genome. In human, the region of exome is about 1% of the genome or approximately 30 million base pairs. There are two steps in whole exome sequencing, first is the selection of only the encoded protein, DNA or exon by using array-base capture or in-solution capture. Then, there is sequencing step. Nowadays, there are many Next Generation Sequencing platforms for sequencing such as Illumina and Ion Torrent. This technique has been applied for clinical diagnosis. In clinical diagnosis, most of the known mutations that causes disease occur in exons. This is why whole exome sequencing has been used for improve precision of clinical diagnosis. For example, it can be used to analyse the whole-exome of the melanoma tumors patients to generate the mutation patterns of individual person and also used to observe the overall mutation load of the melanoma tumors patients for predicting the response to anti-PD-1 therapy of the patient.

2.2. Aj. Chalongrat’s class: Scaffoldless enzyme assembly

Protein assembly by using scaffold have been shown to improve production activity of enzyme group. Scaffold-mediated enzyme complex helps in proximity channeling which makes enzymes come together close enough and the enzymatic product can move to next reaction easily before it diffuses away. However, using scaffold-mediated enzyme complex tends to produce large and disordered multienzyme complex. Scaffoldless supramolecular enzyme complex is another way to make proximity channeling in enzyme complex by using principle of binding of two molecules (ligand and receptor) which are attached with the selected enzyme.

2.3. Aj. Nitwara’s class: QD-labelled real time image analysis

Video microscopy and single-particle tracing of fluorescently labelled molecules allow the motion or dynamics tracking of labelled protein. Normally, only fluorescent dye is not efficient enough for long term imaging. There is one technique that offers an advantage over traditional fluorescent dye which is Quantum Dots (QDs). QDs are semiconductor nanocrystals which is one type of colloids for biolabeling. When comparing the size with GFP, QDs are approximately the same in size as a GFP molecule ,but QDs brightness are 20 times more intense and more photo-stable (Nelson,et al., 2011). QDs have very narrow fluorescence emission spectra, but very broad excitation spectra which allow multi-usability of QDs. A large number of QDs surface coatings are commercially available, which allows for a variety of attachment strategies, both covalent and noncovalent. QDs can be used as a good internal labeling strategy for developing fluorescent viral particles. For example, the Vpr proteins of HIV-1 was conjugated with QDs  to study viral infection in real time and to observe the mechanism of HIV-1 that infects to the macrophages.  

2.4. Aj. Albert’s class: Genetic knockout

        To study gene function, genetic knockout is a technique to control specific gene for study its function. The genetic knockout is usually done in mice model since it shares many genes related to human. To inactivate gene, the target gene is replaced or disrupted with artificial piece of DNA. When the gene was inactivated, we can observe phenotypic changes of mice e.g. appearance, behaviour, physiology and hematological change. Knockout mice can be used to study the gene that might contribute to different kind of human’s disease. For example, knockout mice can be applied to investigate the role of GSTO1-1 in inflammatory pathways by characterizing Gsto1 knockout mice and investigating the response in inflammatory disease models, including inflammatory shock from LPS, dextran sodium sulfate mediated colitis and pro-inflammatory high fat diet. GSTO1-1 deficiency mice show more severe inflammation. GSTO1-1 deficiency mice are resistant to LPS induced inflammatory shock, less susceptible to HFD-induced obesity and inflammation. However, GSTO1-1 deficiency mice increased susceptibility to dextran sodium sulfate induced colitis. So GSTO1-1 plays a critical role in pro-inflammatory response suggested that GSTO1-1 can be used as a new target for the development of novel anti-inflammatory drugs.

2.5. Aj. Poochit’s class: Engineered bacteriophage

Bacteriophages or phages are viruses that invade bacterial cells then disrupt bacterial metabolism and cause the bacteria to die. We have been using antibiotics to kill bacteria for a long time. Nowadays, there are many people who are antibiotic resistant, so bacteriophage is another choice to be used for clinical purposes. Bacteriophage has a specific recognition to bacterial host, so we can use this strategy to kill a particular bacteria instead of using some antibiotic which can kill many bacteria at the same time. Bacteriophage are safer and more precise than using antibiotic drug. Moreover, we can modify bacteriophage to combat the target bacteria by using genetic engineering. For example, phages cocktail containing multiple engineered phages could effectively remove a selected bacterial targets in mixed microbial population.

2.6. Aj. Wipa’s class: Localization of target protein

Fluorescent tag also known as fluorescent label or fluorescent probe, is a molecule that can bind and can be used to detect a biomolecule such as a protein, antibody, or amino acid. Normally, fluorescent tag is a fluorescent molecule known as fluorophore. The fluorophore can bind to a specific region on protein, antibody, or amino acid.  The use of fluorescent tag can be applied for cell imaging with fluorescent microscopy that allows the visualization of specific proteins. We can study cellular biology such as the localization and functions of proteins in cellular membranes and organelles by tagging the target proteins with fluorescent tag. For example, CrLAAT1 was tagged with GFP protein to study the localization of CrLAAT1 in microalgae and the result showed that  CrLAAT1 was targeted to chloroplast.

2.7. Aj. Duncan’s class : Making an Artificial Data and Result in Scientific Publication

There are many researchers who did an artificial data to make a story to convince the readers. They use image manipulation to make a figure in their publication. For example, they take one picture ,but use this figure in many aspects such as rotation, deletion of some part of the picture, reflection and picture enlargement. For the gel or Western blot picture which showed band, they copy and paste an artificial band to match their expected or designed results. Another method, they copy a result from past publication to their publication. Nowadays, journal publishers know this problem, so they decided to have a forensic sesion to detect any fake picture or graph in publication. This is a lesson for us to carefully think about the figures when we look at them and try to not 100% trust the results from scientific publication. We have to think about the logic, reason and possibility of the reported result.

2.8. Aj. Apinunt’s class: Plagiarism checker software

        Plagiarism is one of the most concern issues in the scientific research and publications mostly done by copying language and ideas from another author. Although, plagiarism in some contexts is considered as theft or stealing, some people still do it. Therefore, we can identify plagiarism in publication by plagiarism detection software. For example, Turnitin which can be accessed at https://www.turnitin.com/en_us/home. Turnitin will compare similarity of the submitted work to database including data on the internet, published works and student papers. It supports more than 30 languages including English and Thai. For Mahidol students, lecturers and staffs, we can request for account ID and enrollment password to use Turnitin at
http://www.li.mahidol.ac.th/account-turnitin/.

3. Conclusion

Molecular biology is the study of biology at a molecular level, and is not so much considered as a technique as an approach. It needs knowledge of basic sciences. Researchers in molecular biology use specific techniques native to molecular biology. The Current Topic class in the year 2018 shows that there is an increasing development of molecular biology techniques in the present which helps to open the worldview on our study. The progress of technology in molecular biology have to be fast, comfortable, and save time for researchers’ work. However, it causes a highly competitive environment and pressure for researcher to publish papers that causes the problem in scientific community. The methodology in scientific publication is necessary for researchers in the next-generation. Thereby, authors should not write too much in details and make it easy to understand. To date, there are many problems in published scientific journals. Most background information in publication is ancient, so it is difficult to follow. Some published papers are retracted because they have some fake  picture or graph in the results part of the publication. Therefore, we as researcher should carefully think about the results from picture or graph in the scientific publication. We should not completely trust the result that maybe not be the truth, so we have to think about the common sense, logic, reason and possibility of the experiment results. One of the problems that scientific researchers should mostly concern in scientific publication is plagiarism in research.

 

4. References

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3.          Khadilkar SS. The plague of plagiarism: prevention and cure!!! The Journal of Obstetrics and Gynecology of India. 2018;68(6):425-31.

4.          Li D, Tomljenovic L, Li Y, Shaw CA. RETRACTED: Subcutaneous injections of aluminum at vaccine adjuvant levels activate innate immune genes in mouse brain that are homologous with biomarkers of autism.J. Inorg. Biochem. 2017;177:39-54.

5.         Li Q, Li W, Yin W, Guo J, Zhang Z-P, Zeng D, et al. Single particle tracking of human immunodeficiency virus type 1 productive entry into human primary macrophages. 2017.

6.             Menon D, Innes A, Oakley AJ, Dahlstrom JE, Jensen LM, Brüstle A, et al. GSTO1-1 plays a pro-inflammatory role in models of inflammation, colitis and obesity. Sci. Rep. 2017;7(1):17832.

7.          Price JV, Chen L, Whitaker WB, Papoutsakis E, Chen W. Scaffoldless engineered enzyme assembly for enhanced methanol utilization. Proceedings of the National Academy of Sciences. 2016.

8.         Yamaoka Y, Achard D, Jang S, Legéret B, Kamisuki S, Ko D, et al. Identification of a chlamydomonas plastidial 2-lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content. Plant Biotechnol. J. 2016;14(11):2158-67.

9.        Nelson SR, Ali MY, Warshaw DM. Quantum dot labeling strategies to characterize single-molecular motors. Methods Mol Biol (Clifton, NJ). 2011;778:111-21

10.        Siu K-H, Chen RP, Sun Q, Chen L, Tsai S-L, Chen W. Synthetic scaffolds for pathway enhancement. Curr Opin Biotechnol. 2015;36:98-106.

11.        Sulakvelidze A, Alavidze Z, Morris JG, Jr. Bacteriophage therapy. Antimicrob Agents Chemother. 2001;45(3):649-59.