3. Tenascin–C (TN-C)
According to the cellular structure of the liver, the perisinusoidal space (or space of Disse) is the location lies between the hepatic cords and sinusoids of the liver. It is commonly filled with various extracellular matrix (ECM) proteins whose function is to support the hepatic cords, regulate the proliferation and functions of hepatocytes and maintain the integrity of liver. CITATION Tan06 l 1033 (Tanaka, et al., 2006). When the liver is exposed to an acute injury, it can restore its original architecture in a relatively short period even when a large part of the organ is destroyed. On the other hand, chronic liver injury induces repetitive tissue damage, resulting in disabled regenerative capacity with inflammatory infiltrate and a chronic wound healing response. The response to chronic injury also includes necrosis and/or apoptosis of parenchymal cells and their replacement by extracellular matrix (ECM)CITATION Bat05 l 1033 (Bataller, 2005).

The tenascins are a highly conserved family of large oligomeric glycoproteins found in the extracellular matrix (ECM) of vertebrate organisms. The family contains four family members: tenascin-X, tenascin-R, tenascin-W, and tenascin-C.CITATION Bre09 l 1033 (Brellier, Tucker, & Chiquet-Ehrismann, 2009) CITATION Chi111 l 1033 (Chiquet-Ehrismann & Tucker, 2011). Tenascin-C (TN-C) is a hexameric multimodular ECM glycoprotein that is encoded by the TN-C gene in humans and is mostly expressed during embryonic development and tissue repair in adults and intensively induced by inflammation and cancer.CITATION Kas15 l 2057 (Kasprzycka, Hammarström, & Haraldsen, 2015) Tenascin-C has several molecular forms generated through alternative splicing and protein modifications. (Midwood & Orend, 2009) CITATION Mid16 l 1033 (Midwood, Chiquet, Tucker, & Orend, 2016).

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3.1. Molecular structure of tenascin-C
Tenascin-C is an oligomeric extracellular matrix glycoprotein whose molecular weight is approximately 210–400 kDa and is composed of six subunits. The size of tenascin-C subunit varies as a result of an alternative splicing of the fibronectin repeats at the pre-mRNA level. Each subunit contains a Tenascin assembly (TA) domain that forms a coiled-coil at the N-terminus, epidermal growth factor (EGF)-like repeats, fibronectin type III – like (FNIII) repeats, and a fibrinogen-like domain. CITATION Tan06 l 1033 (Tanaka, et al., 2006). Each of these domains interacts with different binding partners, including cell surface receptors and other extracellular components, CITATION Ore05 l 1033 (Orend, 2005). The FNIII repeats contain a region undergoes alternative splicing, which generates various forms with different molecular weights. A small molecular weight variant of TN-C formed after splicing exists constitutively in normal tissues, whereas the large molecular weight variants, including different combinations of alternative spliced FNIII repeats, are particularly expressed in tissues suffering pathological situations CITATION Ghe01 l 1033 (Ghert, et al., 2001) CITATION Has04 l 1033 (Hasegawa, et al., 2004) CITATION Tan06 l 1033 (Tanaka, et al., 2006)

Fig.1 Multidomain structure of human tenascin-C (TN-C) subunit. The amino-termini of six TN-C subunits are united to construct a hexamer. Each subunit consists of 14+1/2 epidermal growth factor-like domains, 8–15 fibronectin type III (FNIII) domains, depending on alternative RNA splicing, and a single fibrinogen-like domain. The universal FNIII domains (FNIII repeats 1–5 and FNIII repeats 6–8) are present in all TN-C variants. CITATION ElK07 l 2057 (El-Karef, et al., 2007)3.2. Role of tenascin-C in liver injury
Obviously, inflammation is the immediate and serious response to tissue injury and infection. Tenascin-C is induced and expressed at sites of inflammation regardless of the location or type of causative agent. In case of liver injury, TN-C is upregulated and accumulated at parenchymal-connective tissue interfaces and in perisinusoidal spaces and its expression is concentrated in areas of increased immune cell infiltration. In addition, TN-C promotes activation of T lymphocytes that secrete inflammatory cytokines including IFN-?, TNF-?, and IL-4, which in turn activate quiescent hepatic stellate cells (HSCs) that differentiate into myofibroblasts and secrete large amounts of extracellular matrix proteins especially type I collagen (Col I) CITATION Tro12 l 2057 (Troeger, et al., 2012) CITATION Dep15 l 2057 (Depito & Schwabe, 2015).
Generally, all tenascins have the capacity to alter cell adhesion directly or via an interaction with fibronectin and cell-tenascin interactions lead to increased cell motility. For TN-C, there is a correlation between its elevated expression and increased cell metastasis as it retards cell attachment and dispersion on ECM proteins (eg: fibronectin and laminin) and stimulates cell tethering and rolling. CITATION Cla97 l 2057 (Clark, Erickson, & Springer, 1997) CITATION Brö13 l 2057 (Brösicke, van Landeghem, Scheffler, & Faissner, 2013). Previous studies conducted on mice suffering concanavalin A-induced hepatic fibrosis revealed that mice lacking TN-C exhibited reduced inflammatory cell and myofibroblast infiltration with a sharp decline in inflammatory cytokine expression. Moreover, transforming growth factor-? (TGF)-? mRNA was upregulated in wild type mice and was significantly higher than its level in TN-C null mice. CITATION ElK07 l 2057 (El-Karef, et al., 2007). Consequently, it was potentially suggested that there is a contribution between TN-C and progression of liver fibrogenesis.

Observations on previous studies on mice:
1. Gradual increase in TNC deposition in WT mice compared with TNKO mice.

2. TNC mRNA levels increase sequentially and peaked after the 9th injection in WT mice.

3. Collagen deposition was less intense in TNKO than WT mice.

4. Procollagen I and III transcripts were significantly upregulated in WT compared with TNKO mice.

5. Inflammatory infiltrates were most prominent after the 3rd-6th injections in both groups and were less intense in TNKO than WT mice.

6. Interferon gamma, tumor necrosis factor alpha and interleukin-4 mRNA levels were significantly higher in WT than TNKO mice.

7. Activated hepatic stellate cells (HSCs) and myofibroblasts (cellular source of TNC and procollagens) were more common in WT livers.

8. Transforming growth factor-beta (TGF-?1) mRNA expression was significantly up regulated in WT type but not in TNKO mice.


3. Above average returns- In order to earn above average returns, your business must have a competitive advantage over your rivals. In order to achieve an above average return, you must develop a competitive strategy. This means developing a strategy for how you are going to compete in the market place by cost-leadership means that you are the cheapest option every time or differentiation means that your product or service is different from others on offer which allows you to charge a premium price. To make a cost-leadership strategy work you would offer products of average quality for less than the average price. If you are the only one doing that, and you can actually produce at a lower cost, then you may have a competitive advantage and earn those above average returns. Example: making a product that is cheaper in terms of cost and manufacturing but just as good/better in terms of quality & sensory over your competitors
Strategic competitiveness is a type a strategy that certain firm can plan to achieve their organizational goals even though there are a lot of competitors around them. It can be achieved when a certain company or firm successfully come out with a special ideas or strategy that can allows the firm to create wealth to its organization when it is implemented or in other word, implementing value-creating strategy. Usually, in implementing strategic competitiveness, other companies are unable to duplicate or follow it. Some even feels that it is too costly to follow.


3.1 Cell resurrection
Fibroblast and macrophage cells will be taken from Faculty of Medicine, UniSZA. The fibroblast cell are used for the purpose of representing human normal cells while the macrophage cells are for representing human immune cells in the effort to detect the cell response in the body when this compound is introduced. The cells will be resurrected from cyropreservation. The cryovials containing frozen cells will be put into a 37°C water bath for thawing process. The vials will be gently swirled in the 37°C waterbath for less than 1 minute until there is just a small bit of ice in the vial.
Cryovials will be transferred from water bath into a laminar flow hood. The outside of the hood is made sure to be sterilized with 70% ethanol. Thawed cells will be transferred dropwise into a centrifuge tube containing prewarmed media and fetal calf serum.
The cell suspension will then be centrifuged at 200 rpm for 5 minutes. After centrifugation is completed, cells will be transferred into flask gently with DMEM growth medium. The cells will be incubated in 5% CO_2 incubator.

3.2 Cell culture
Preparation of growth medium will be done prior to culturing. 500 ml of pre-made Dulbecco’s Modified Eagle’s media (DMEM) will be supplemented with 50 ml (10%) of fetal calf serum, 100 U/ml penicillin/streptomycin. This media is for culturing fibroblast cells. For the macrophage cells, Roswell Park Memorial Institute medium (RPMI1640) will be used as growth medium. The media powder will be dissolved in 1 L of distilled water in a beaker. While it is stirred on a hot plate, sodium bicarbonate will be added. Then, the pH of the media will be adjusted to 7.4. The RPMI1640 will also be supplemented with 100 ml (10%) of fetal calf serum, and 200 U/ml penicillin/streptomycin.

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Resurrected cells will be passaged into new culture flasks. Two T175 flasks will be filled with growth culture media. Next, cells are taken out from incubator and the used culture media will be disposed. The cells will then be trypsinized with 0.25% trypsin-EDTA solution to detach the cells from the surface of the culture flask. Growth media will be added to stop trypsin reaction with the cells. Then, the total amount of medium in the flask will be splitted into the two new culture flasks and followed with incubation.

Cell maintenance will be done by replacing fresh medium twice every week. Cultures with confluency more than 95 % will be ppassage into new culture flasks while cultures with 75-95 % confluency will be used for the experiment. Cell confluency is to be tested with trypan blue exclusion test. Cells will be seeded in 96 well plate one day prior to treatment of thiosemicarbazide.

3.3 Compound preparation
The thiosemicarbazide compound will be prepared for a stock solution with the concentration of 10 mM by the addition of complete media and DMSO. A serial dilution with varying concentration of 0.1 mM, 0.01 mM, 0.001 mM, 1 X ?10?^4 mM, 1 X ?10?^5 mM, & 1 X ?10?^6 mM will be prepared from the stock.

3.4 Cell viability assay
The cell viability will be assesed by MTT assay where the protocol starts from trypsinization of the cells. This is to achieve the first objective of the study. The cells will be splitted and seeded into 96 well plate. Then, the cell is left until the confluency is 90-100 %, and it will be ready for use. TSC-Ni treatment will be added to the wells according to the desired concentrations with 5 replicates each. Cells will be incubated for 24 hours in 5% CO2 incubator. This is to allow reaction between the compound and the cells to take place.
MTT solutions will be prepared by dissolving 1 mg MTT in 1 mL PBS solution and the molarity will be at 2.41 mM. 20 µL of the solution into each well together with 160 µl of growth media and followed by incubation for 4 hours. After discarding media, 100 µl of DMSO will be added to the well. Then, the well will be covered with aluminium foil and mildly shaked for 15 minutes. The reading of the plate will be done using fluorescent reader in 570 nm absorbance.

To address the second objective, a list of targeted proteins will be screened using Swiss target prediction (www.swisstargetprediction.ch) and a simulation of docking of the proteins with TSC-Ni will be done on Systems dock (systemsdock.unit.oist.jp).

3.5 Assessment on mode of cell death
AO/PI staining protocol will be used to assess mode of cell death which is the third objective. Firstly, cells should be harvested into centrifuge tube. Cells will be removed from incubator and trypsinization will take place to detach the cells. To let the cells react with trypsin, cells will be reincubated for 10 mins. Then, trypsinization will be stopped by adding growth media into the flask. The cells will be centrifuged at 1500 rpm for 5 minutes.

Next, the cells are to be suspended in PBS. In a separate centrifuge tube, aliquot some cell suspension and add AO/PI with ratio 5:1.
AO will stain both viable and unviable cells thus PI stain is used to counterstain the unviable cells, enabling to differentiate between living cells and dead cells. Then, properties of membrane permeability and mode of cell death will be determined.

3.6 Statistical analysis
Analysis of cytotoxic activity will be assessed by using two-tail T test. Number of cell viable cells stained by MTT solution in treated cells will be compared with control (untreated cells), and the reading of absorbance on microplate reader will be taken as the measure of cell viability. results will be expressed as mean +/- standard deviation with each sample being tested in at least triplicate. Plotting a curve with percentage of cell viability (on y-axis) against varying concentration of TSC-Ni (on x-axis) will determine the IC_50 value of the compound. Two-tail T test analysis will be done at 95% significant level.


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