Colin Barrow, Ph.D.
Chair in Biotechnology, School of Life & Environmental Sciences, Deakin University, Australia
Nano-biotechnology: Omega-3 Oils and Nanofibres
The health benefits of long-chain omega-3 fatty acids are well established, especially for eicosapentaenoic acid (EPA) and docosapentaenoic acid (DHA) from fish and microbial sources. In fact, a billion dollar market exists for these compounds as nutritional supplements, functional foods and pharmaceuticals. This presentation will describe some aspects of our omega-3 biotechnology research that are at the intersection of Nano-biotechnology and oil chemistry. These include the use of lipases for the concentration of omega-3 fats, through immobilization of these lipases on nanoparticles, and the microencapsulation and stabilization of omega-3 oils for functional foods. I will also describe some of our work on the enzymatic production of resolvins using lipoxygenases, and the fermentation of omega-3 oils from marine micro-organisms. Finally, I will describe some of our work on the formation of amyloid fibrils and graphene for various applications in nano-biotechnology.
Deepthy Menon, Ph.D.
Associate Professor, Centre for Nanosciences & Molecular Medicine, Health Sciences Campus, Amrita University, Kochi, India
Nanobioengineering of implant materials for improved cellular response and activity
Deepthy Menon, Divyarani V V, Chandini C Mohan, Manitha B Nair, Krishnaprasad C & Shantikumar V Nair
Abstract
Current trends in biomaterials research and development include the use of surfaces with topographical features at the nanoscale (dimensions < 100 nm), which influence biomolecular or cellular level reactions in vitro and in vivo. Progress in nanotechnology now makes it possible to precisely design and modulate the surface properties of materials used for various applications in medicine at the nanoscale. Nanoengineered surfaces, owing to their close resemblance with extracellular matrix, possess the unique capacity to directly affect protein adsorption that ultimately modulates the cellular adhesion and proliferation at the site of implantation. Taking advantage of this exceptional ability, we have nanoengineered metallic surfaces of Titanium (Ti) and its alloys (Nitinol -NiTi), as well as Stainless Steel (SS) by a simple hydrothermal method for generating non-periodic, homogeneous nanostructures. The bio- and hemocompatibility of these nanotextured metallic surfaces suggest their potential use for orthopedic, dental or vascular implants. The applicability of nanotextured Ti implants for orthopedic use was demonstrated in vivo in rat models, wherein early-stage bone formation at the tissue-implant interface without any fibrous tissue intervention was achieved. This nanoscale topography also was found to critically influence bacterial adhesion in vitro, with decreased adherence of staphylococcus aureus. The same surface nanotopography also was found to provide enhanced proliferation and functionality of vascular endothelial cells, suggesting its prospective use for developing an antithrombotic stent surface for coronary applications. Clinical SS & NiTi stents were also modified based on this strategy, which would offer a suitable solution to reduce the probability of late stent thrombosis associated with bare metallic stents. Thus, we demonstrate that nanotopography on implant surfaces has a critical influence on the fate of cells, which in turn dictates the long term success of the implant.
Acknowledgement: Authors gratefully acknowledge the financial support from Department of Biotechnology, Government of India through the Bioengineering program.
D. Narasimha Rao, Ph.D.
Professor, Dept of Biochemistry, Indian Institute of Science, Bangalore, India
Genomics of Restriction-Modification Systems
Restriction endonucleases occur ubiquitously among procaryotic organisms. Up to 1% of the genome of procaryotic organisms is taken up by the genes for these enzymes. Their principal biological function is the protection of the host genome against foreign DNA, in particular bacteriophage DNA. Restriction-modification (R-M) systems are composed of pairs of opposing enzyme activities: an endonuclease and a DNA methyltransferase (MTase). The endonucleases recognise specific sequences and catalyse cleavage of double-stranded DNA. The modification MTases catalyse the addition of a methyl group to one nucleotide in each strand of the recognition sequence using S-adenosyl-L-methionine (AdoMet) as the methyl group donor. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, R-M systems are generally classified into three groups. In general R-M systems restrict unmodified DNA, but there are other systems that specifically recognise and cut modified DNA. More than 3500 restriction enzymes have been discovered so far. With the identification and sequencing of a number of R-M systems from bacterial genomes, an increasing number of these have been found that do not seem to fit into the conventional classification.
It is well documented that restriction enzyme genes always lie close to their cognate methyltransferase genes. Analysis of the bacterial and archaeal genome sequences shows that MTase genes are more common than one would have expected on the basis of previous biochemical screening. Frequently, they clearly form part of a R-M system, because the adjacent open reading frames (ORFs) show similarity to known restriction enzyme genes. Very often, though, the adjacent ORFs have no homologs in the GenBank and become candidates either for restriction enzymes with novel specificities or for new examples of previously uncloned specificities. Sequence-dependent modification and restriction forms the foundation of defense against foreign DNAs and thus RM systems may serve as a tool of defense for bacterial cells. RM systems however, sometimes behave as discrete units of life, and any threat to their maintenance, such as a challenge by a competing genetic element can lead to cell death through restriction breakage in the genome, thus providing these systems with a competitive advantage. The RM systems can behave as mobile-genetic elements and have undergone extensive horizontal transfer between genomes causing genome rearrangements. The capacity of RM systems to act as selfish, mobile genetic elements may underlie the structure and function of RM enzymes.
The similarities and differences in the different mechanisms used by restriction enzymes will be discussed. Although it is not clear whether the majority of R-M systems are required for the maintenance of the integrity of the genome or whether they are spreading as selfish genetic elements, they are key players in the “genomic metabolism” of procaryotic organisms. As such they deserve the attention of biologists in general. Finally, restriction enzymes are the work horses of molecular biology. Understanding their enzymology will be advantageous to those who use these enzymes, and essential for those who are devoted to the ambitious goal of changing the properties of these enzymes, and thereby make them even more useful.
V. Nagaraja Ph.D.
Professor, Indian Institute of Science, Bengaluru, India
Perturbation of DNA topology in mycobacteria
To maintain the topological homeostasis of the genome in the cell, DNA topoisomerases catalyse DNA cleavage, strand passage and rejoining of the ends. Thus, although they are essential house- keeping enzymes, they are the most vulnerable targets; arrest of the reaction after the first trans-esterification step leads to breaks in DNA and cell death. Some of the successful antibacterial or anticancer drugs target the step ie arrest the reaction or stabilize the topo -DNA covalent complex. I will describe our efforts in this direction – to target DNA gyrase and also topoisomerase1 from mycobacteria. The latter, although essential, has no inhibitors described so far. The new inhibitors being characterized are also used to probe topoisomerase control of gene expression.
In the biological warfare between the organisms, a diverse set of molecules encoded by invading genomes target the above mentioned most vulnerable step of topoisomerase reaction, leading to the accumulation of double strand breaks. Bacteria, on their part appear to have developed defense strategies to protect the cells from genomic double strand breaks. I will describe a mechanism involving three distinct gyrase interacting proteins which inhibit the enzyme in vitro. However, in vivo all these topology modulators protect DNA gyrase from poisoning effect by sequestering the enzyme away from DNA.
Next, we have targeted a topology modulator protein, a nucleoid associated protein(NAP) from Mycobacterium tuberculosis to develop small molecule inhibitors by structure based design. Over expression of HU leads to alteration in the nucleoid architecture. The crystal structure of the N-terminal half of HU reveals a cleft that accommodates duplex DNA. Based on the structural feature, we have designed inhibitors which bind to the protein and affect its interaction with DNA, de-compact the nucleoid and inhibit cell growth. Chemical probing with the inhibitors reveal the importance of HU regulon in M.tuberculosis.