Component 8 Research & Innovation Colleges
Mass propagation of candidate lead compound(s)using Plant systems
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Mass propagation of candidate lead compound(s)using Plant systemsAnti cancer activity of candidate lead compound(s)using cell lines List of identified anti-QS and anti-biofilmagents 
Antibacterial activity Antibacterial activity of the edible source was assessed against bacterial pathogens like S. aureus, S. aureus MRSA, Bacillus subtilis, E. coli, P. aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, P. vulgaris, Shigella dysenteriae, S. flexneri, S. sonnei, S. boydii and Aeromonas hydrophila. Significant antibacterial activity was observed only against Gram positive pathogens like S. aureus, S. aureus MRSA, B. subtilis (Figure 1) and negligible activity was found against P. mirabils, P. vulgaris and A. hydrophila. Since methanol extract showed maximum activity among the three extracts (acetone, ethyl acetate and methanol), it was chosen for all furtherexperiments.
Table 1: Antibacterial activity of various solvent extracts of ediblesource. 
S. aureus MRSA B. subtilis Figure 1: Antibacterial activity shown by the methanolic extract of ediblesource against Gram positive bacteria. Antibiofilm activity Antibiofilm activity was tested with the bioactive molecule against the same battery of microbes used for antibacterial activity testing. Antibiofilm assay was done by allowing the bacteria to grow on a glass cover slip in the presence and absence of the extract in a 24 well microtitre plate as stated by Bakkiyaraj & Pandian (2010). Then the slides were subjected to Z-stack analysis in a Confocal Laser Scanning Microscope (Zeiss LSM 710, Carl Zeiss, Germany) and image analysis was done with Zen 2009 software as described earlier (Bakkiyaraj & Pandian, 2010). Methanolic extract of edible source was found to inhibit the biofilms of most of the pathogens which was evidenced from the images given in Figure 2. Z-stack analysis showed the reduction in the thickness of the biofilm in treated samples compared to their untreated controls.
µg/ml for E. coli and 250 µg/ml for C. albicans (Figure 3). 
Figure 3. Effect of extract on biofilm formation. 3a. Effect of extract on bacterial biofilm formation was studied and found that a minimum concentration of 125 µg/ml of extract is required for the inhibition of biofilms of S. aureus and MRSA. Similarly, 150 µg/ml for E. coli biofilms. 3b. BIC for C. albicans was found to be 250 µg/ml, above which there is no significant change in biofilm inhibition. (* represents the significance (p value < 0.05)) Further, the ability of the extract to disrupt the pre-formed biofilms of S. aureus, MRSA, E. coli and C. albicans was tested at their BIC. It was found that pre-formed biofilms of S. aureus, MRSA, E. coli and C. albicans were significantly disrupted in both nutrients limited (supplemented with PBS + PME) as well as nutrient enriched (supplemented with media + PME) conditions (Figure 4). Biofilm disruption was found to be around 70% each for S. aureus, MRSA, E. coli and 90% for C. albicans.
Folin-Ciocalteau method is a specific assay that was followed for the quantification of total phenols (monohydric phenols, polyphenols, flavonoids and tannins). The evaluation of total phenols shows that the extract contained one mg of phenolic compounds per gram of the dry weight of edible source. To get more insight regarding the nature of phenolic compounds present in the extract, HP-TLC analysis was done. Since, several reports demonstrated the presence of tannins, chiefly ellagitannins to be the major bioactive lead in the extract, bioactive molecule X (name of the bioactive molecule is undisclosed to maintain confidentiality) was used as a standard for HP-TLC analysis (Adams et al., 2006; Glazer et al., 2012). HP-TLC analysis of the extract has substantiated the presence of bioactive molecule X as a major component and 8 other compounds in trace amounts (Table 1 and Figure 5a & b). The major peak (peak
X 1 0.44 264.1 11098.4 bioactive molecule Sample A - represents extract and ELG - represents the standard ellagic acid. Antibacterial and antibiofilm activities of bioactive moleculeX Antibacterial and antibiofilm activities of bioactive molecule X were assessed against S. aureus, MRSA, E. coli and C. albicans. Higher concentrations (>75 - 100 µg/ml) have shown antibacterial and antifungal activity (Figure 6) and hence the antibiofilm activity of bioactive molecule X was assessed at sub-lethal concentrations as less as 5 – 40 µg/ml. Sub-lethal concentrations (<40 µg/ml) i.e. ½ MIC or less than that have shown dose dependent biofilm inhibition against all the test pathogens (Figure 7). This study thus confirms that the antibiofilm activity ofextract is because of its bioactive molecule X
Understanding the mode of action and interaction between bioactive molecule X and host proteins will offer valuable information with which highly efficient drugs could be developed in near future. Hence, the protein were isolated and analysed through both 1D and 2D Gel electrophoresis. Two Dimensional - Poly Acrylamide Gel Electrophoresis(2D-PAGE) Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was introduced by O’Farrell and Klose in 1975, and it still remains as a gold standard technique to separate complex protein mixtures. 2D- PAGE based proteomics work flow includes preparation of protein sample, isoelectric-focusing [IEF] of proteins with immobilized pH gradients (IPGs) based on isoelectric pH, reduction, alkylation, separation according to an apparent molecular weight, visualization of protein spots, imaging and in gel tryptic digestion of proteins for mass spectrometry (MS)-driven identification. The uniqueness of this technique is easy visualisation of protein isoforms and the ability to resolve more than 1800 proteins in a single gel. Introduction of highly sensitive, wide dynamic range of protein detection and mass spectrometry compatible CyDyes based difference gel electrophoresis (DIGE) overcomes gel to gel variations a major concern of 2D-PAGE. The presence of Quorum Sensing (QS) systems, formation of highly protective biofilm and the emergence of multiple antibiotic resistance in an alarming number of human pathogens has necessitated traditional microbiological research into proteomics based microbial pathogenicity research. Gel based proteomic analysis recently reveals the pathoproteome and several immunogenic proteins of Pseudomonas aeruginosa, Candida albicans and Chlamydia trachomatis. Chemicals
Reagents Urea and Thiourea (UT) sample preparation buffer
*Added prior to use. Stored in 2.5-ml aliquots at -20 °C. Urea and Thiourea (UT) rehydration buffer
*Added prior to use. Stored in 2.5-ml aliquots at -20 °C. SDS equilibration buffersolution
*The stock solution was stored at -20 °C in 20-ml aliquots. Just prior to use, 1% of DTT and 2.5% of iodoacetamide was for first and second equilibration, respectively. 10x Laemmli SDS electrophoresis buffer
Stored at room temperature. 30% acrylamide, 0.8% N, N-methylenebisacrylamide monomersolution
Stored at 4 °C in the dark 4x resolving gel buffer solution
Stored at 4 °C. Bromophenol blue stock solution
12.5% Acrylamide gel
Agarose sealing solution
Extracellular protein preparation overnight culture of P. aeruginosa ATCC10145 was inoculated in 100 mL LB broth and incubated it for 24 hrs in the presence and absence of bioactive molecule X. After incubation, the culture was transferred to 50 ml falcon tube and it was centrifuges at 10,000xg for 15 minutes. The supernatant was filtered in 0.22 µm pore size membrane filter to remove bacterial cells. 0.2 mg/mL of deoxycholic acid and it was incubated in ice for 30 minutes. 10% of TCA was added and incubated overnight for protein precipitation. The tube was centrifuged at 12,000 x g for 30 minutes and the supernatant was discarded. The pellet was resuspended with small volume of Milli Q and it was precipitated again with 8 volumes of ice-cold acetone at 4°C for 2 hours. After centrifugation, the pellet was air dried at room temperature for 5 minutes. The pellet was thoroughly dissolved in an appropriate volume of solubilization buffer and centrifuged at 50,000 x g for 40 minutes at 10°C to remove insoluble materials. Phenol Chloroform extraction was done and to the proteins present in the lower organic phase was precipitated with 5 volumes of ice-cold acetone. The protein was recovered and it was suspended in solubilization buffer. The proteins were quantified using the Bio-Rad protein assay kit (Bio-Rad). 150µg of protein was cleaned as per the manufacturer’s instructions. Passive rehydration of IPG strips and focussing The IPG strips were thawed at room temperature for 30 minutes. 50µl of sample buffer containing 150µg of extracellular protein was added to the UT rehydration buffer and it was incubated at room temperature for 30 minutes. The rehydration solution containing protein sample was transferred to the reswelling tray. The IPG strips were allowed to rehydrated overnight at room temperature. Isoelectric focussing was done using IPGphor (GE Health care) at 20°. The IPG strips was focussed for 19 hrs by following the scheme.
Equilibration The focussed strips was placed in rehydration tray (gel side facing up) containing SDS- equilibration Buffer 1 (1% DTT and trace amount of Bromophenol blue) and incubated at room temperature for 20 minutes with gentle rocking. The strips were transferred to SDS-equilibration buffer 2 (2.5% iodoacetamide and trace amount of Bromophenol blue) and incubated at room temperature for 20 minutes with gentle rocking. Second Dimension The glass plates were cleaned with double distilled water and he plates were wiped with alcohol. 500 ml of acrylamide gel mix was prepared and it was poured inside the gel cassettes at a slow flow rate without introducing air bubbles.The gels were overlaid with 0.1% SDS solution and were allowed to polymerize at room temperature for 2 hours. The multiple gel caster with polymerized gels was transferred to cold room (4°C) for overnight aging. After equilibrating, the gel plates were fixed in DALT Six tank and the following electrophoretic conditions were used
After the run, the gels were removed from the cassettes and it was placed in fixative solution for 4 to 12 hours for silver staining to visualize the proteins. Silver staining protocol
Figure 8. Effect of bioactive molecule X on the extracellular proteome of MRSA ATCC 33591 The effect of bioactive molecule X on the extracellular proteome was first analyzed by isolating the extracellular proteome of MRSA ATCC 33591 control and treated (Figure 8). It was observed that, most of the proteins were downregulated (box and arrow shown in red) and some of the proteins were upregulated (arrow shown in green). Hence it was evident that, bioactive molecule X had its direct effect on the proteome of MRSA. In order to know further, 2D gel electrophoresis was carried out and standardization is in progress for the extracellular proteome of MRSA treated with bioactive molecule X. In the mean time, the effect of another bioactive molecule Y (name undisclosed to maintain confidentiality) was analysed for its effect on the intracellular proteome of Serratia marcescens (Figure 9). Based on the results obtained in 2D gel electrophoresis, it was observed that, most of the proteins were downregulated when treated with bioactive molecule Y. This directly implies that further evaluation of the downregulated protein will throw more lights on the possible mechanism of action of the bioactive molecule Y on its anti-biofilm activity. Identification of the downregulated proteins using MALDI TOF-TOF (Schimadzu, Axima Performance, Germany) is under progress. Figure 9. Effect of bioactive molecule Y on the extracellular proteome of Serratia marcescens (red colour box-downregulated proteins; Green box-upregulated proteins) In vivo analysis of the effect of bioactive molecule using Caenorhabditis elegans as model organism The effect of bioactive molecule on in vivo biofilm formation by S. aureus, MRSA and SA-CI-18 was assessed on the model organism nematode C. elegans using a method modified from Jansen et al. (2002). C. elegans in batches of 10 animals for each bacterial strain was taken in a 24-well polystyrene plate containing TSB with bioactive molecule, and wells without bioactive molecule extract acted as controls. Wells were inoculated with 1% (1 x103 CFU ml-1) of S. aureus, MRSA and SA-CI- 18 respectively from log phase cultures and incubated for 24 h at 378C. After incubation an animal from each well was taken, washed three times with M9 buffer and stained with 0.1% acridine orange. The animals were then observed under CLSM and their intestinal colonisation was measured by Z-stack analysis. The intensity of the dye was directly proportional to the amount of bacterial biofilm in the intestine. The effect of bioactive molecule on the in vivo intestinal colonisation of C. elegans was measured crudely by comparing the intensities of colonised intestines of C. elegans in both treated and control samples after staining it with 0.1% acridine orange. The intestines of the nematodes were observed by CLSM Z-stack analysis through the process of optical sectioning. The intensity profiles of control animals infected with S. aureus, MRSA and SA- CI-18 were around 150 units each and those for treated animals were approximately <50 units each. The bacterial load in the intestine was also measured by crushing the animals and plating on TSA after dilution. The bacterial load in the intestine of the control and the bioactive molecule treated C. elegans were 4.3 x103 CFU ml71 and 1.4 x 102 CFU m-1 respectively. These results clearly reflect the ability of bioactive molecule to inhibit the in vivo colonisation of S. aureus in C. elegans to 70% (Figure 10).
Transgenic hairy root cultures derived through genetic transformation of plants with Agrobacterium rhizogenes have revolutionized the role of plant tissue culture in secondary metabolite production. Hairy roots generated from transformed plants have been found to be suitable for the production of wide range of secondary metabolites because of their stable and high productivity in hormone-free culture conditions. They are unique in their genetic and biosynthetic stability, faster in growth, and more easily maintained. Using this methodology, a wide range of chemical compounds has been synthesized. Solanum trilobatum L. (Solanaceae) is considered as one of the most esteemed medicinal plants among Ayurveda and Siddha medical practitioners in India over several decades. Globally the plant is distributed in Indo-Malaysia region with a habit of thorny shrub. It is classified as a Kayalpa (rejuvenator) as it revitalizes the body as well as mind and improves the longevity. Based on the medicinal importance, high frequency genetic transformation system for S. trilobatum L. and the hairy roots were established successfully in liquid medium (Figure11).
In vitro screening of anticancer potential of isolated bioactive molecules using cancer cell lines as a model The anti-cancer potential of the bioactive molecule was assessed using human lung adenocarcinoma cell line A549. 
Figure 12. Phase contrast Microscope image analysis of A549 cells. Antiproliferative activity of bioactive molecule against human lung adenocarcinoma cell line A549 .
Based on the results obtained above, it was clearly understood that the treated group shown shrinked cell morphology with echinoid spikes that directly implied the cytotoxic effect of bioactive molecule against cancer cells in contrast to the control group showing normal cell morphology (Figure 12). In order to know the apoptotic effect of the bioactive molecule, AO/EtBr staining and Fluorescent microscope image analysis of A549 was carriedout.    
Figure 13. AO/EtBr staining and Fluorescent microscope image analysis ofA549 cells. (a). Control (b). A549 cells treated with bioactivemolecule It was observed that A549 cells with green fluorescence in control (a) indicated normal healthy viable cells whereas A549 cells with yellowish green fluorescence in treated cells (b) implied that the cancer cells have undergone apoptosis (Figure 13). Further analysis of the mechanism of action of the bioactive molecule is under progress Annexure II Brief CVs of participating Principal Investigators
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