Hideaki Nagase, Ph.D.
Kennedy Institute of Rheumatology-Centre for Degenerative Diseases, University of Oxford, UK
Osteoarthritis: diagnosis, treatment and challenges
Hideaki Nagase1, Ngee Han Lim1, George Bou-Gharios1, Ernst Meinjohanns2 and Morten Meldal3
- Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, London, W6 8LH UK
- Carlsberg Laboratory, Copenhagen, Denmark,
- Nano-Science Center, Department of Chemistry, University of Copenhagen, Denmark
Osteoarthritis (OA) is the most prevalent age-related degenerative joint disease. With the expanding ageing population, it imposes a major socio-economic burden on society. A key feature of OA is a gradual loss of articular cartilage and deformation of bone, resulting in the impairment of joint function. Currently, there is no effective disease-modifying treatment except joint replacement surgery. There are many possible causes of cartilage loss (e.g. mechanical load, injury, reactive oxygen species, aging, etc.) and etiological factors (obesity, genetics), but the degradation of cartilage is primarily caused by elevated levels of active metalloproteinases. It is therefore attractive to consider proteinase inhibitors as potential therapeutics. However, there are several hurdles to overcome, namely early diagnosis and continuous monitoring of the efficacy of inhibitor therapeutics. We are therefore aiming at developing non-invasive probes to detect cartilage degrading metalloproteinase activities.
We have designed in vivo imaging probes to detect MMP-13 (collagenase 3) activity that participates in OA by degrade cartilage collagen II and MMP-12 (macrophage elastase) activity involved in inflammatory arthritis. These activity-based probes consist of a peptide that is selectively cleaved by the target proteinase, a near-infrared fluorophore and a quencher. The probe’s signal multiplies upon proteolysis. They were first used to follow the respective enzyme activity in vivo in the mouse model of collagen-induced arthritis and we found MMP-12 activity probe (MMP12AP) activation peaked at 5 days after onset of the disease, whereas MMP13AP activation was observed at 10-15 days. The in vivo activation of these probes was inhibited by specific low molecule inhibitors. We proceeded to test both probes in the mouse model of OA induced by the surgical destabilization of medial meniscus of the knee joints. In this model, degradation of knee cartilage is first detected histologically 6 weeks after surgery with significant erosion detectable at 8 weeks. Little activation of MMP12AP was detected, which was expected, as macrophage migration is not obvious in OA. MMP13AP, on the other hand, was significantly activated in the operated knee at 6 weeks compared with the non-operated contralateral knee, but there were no significant differences between the operated and sham-operated knees. At 8 weeks, however, the signals in the operated knees were significantly higher than both the contralateral and sham-operated controls. Activation of aggrecanases and MMP-13 are observed before structural changes of cartilage. We are therefore currently improving the MMP-13 probe for earlier detection by attaching it to polymers that are retained in cartilage.
Ajith Madhavan
Assistant Professor, School of Biotechnology, Amrita University
Development of a Phototrophic Microbial Fuel Cell with sacrificial electrodes and a novel proton exchange matrix
If micro organisms can solve Sudoku and possibly have feelings, who is to say that they cannot also solve the planet’s energy crisis? Mr. Madhavan employs micro organisms to produce energy using microbial fuel cell (MFC). Micro organisms go through a series of cycles and pathways in order to survive, including the Electron Transport Pathway (ETP) in which bacteria release electrons which can be tapped as energy. In a two-chambered MFC, micro organisms interact with an anode in one chamber and in the presence of an oxidizing agent in the cathodic chamber scavenges electrons from the cathode. The two chambers are connected by an external circuit and connected to a load. In between the two chambers is a proton exchange membrane (PEM) which transports protons from the second chamber to the first and acts as a barrier for electrons. Therefore, a renewable source of energy can be maintained by just providing your bacterial culture with the proper nutrients to thrive and remain happy and satisfied (assuming they have emotions).
Mr. Madhavan has done extensive work on such MFCs and has experimented with various micro organisms and substrates to achieve high energy production. The phototropic MFC Mr. Madhavan designed using Synechococcus elongates using waste water as a substrate was able to generate approximately 10 mȦ and 1 volt of electricity. Other research in this area has even shown that using human urine can be used as a substrate for certain bacteria to produce enough energy to charge a mobile phone.
Although this microbial technology seems to be the “next big thing” (despite their small size) when it comes to renewable energy sources there is still a lot of work to be done before these bacteria batteries hit the market. As of now the MFCs are still much less efficient than solar cells and the search for the perfect bacteria and substrate continues.
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.
![Delegate Talk: Intrinsic modulation of cytokine response by mycobacteria @ Acharya Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/btindia.jpg)
Sukhithasri V, Nisha N, Vivek V and Raja Biswas
The host innate immune system acts as the first line of defense against invading pathogens. During an infection, the host innate immune cells recognize unique conserved molecules on the pathogen known as Pathogen Associated Molecular Patterns (PAMPs). This recognition of PAMPs helps the host mount an innate immune response leading to the production of cytokines (Akira et al. 2006). Peptidoglycan, one of the most conserved and essential component of the bacterial cell wall is one such PAMP. Peptidoglycan is known to have potent proinflammatory properties (Gust et al. 2007). Host recognize peptidoglycan using Nucleotide oligomerization domain proteins (NODs). This recognition of peptidoglycan activates the NODs and triggers downstream signaling leading to the nuclear translocation of NF-κB and production of cytokines (McDonald et al. 2005). Pathogenic bacteria modify their peptidoglycan as a strategy to evade innate immune recognition, which helps it to establish infection in the host. These peptidoglycan modifications include O-acetylation and N-glycolylation of muramic acid and N-deacetylation of N-acetylglucosamine (Davis et al. 2011). Modification of mycobacterial peptidoglycan by N-glycolylation prevents the catalytic activity of lysozyme (Raymond et al. 2005). Additionally, mycobacterial peptidoglycan is modified by amidation for unknown reasons.
Here, we have investigated the role of amidated peptidoglycan in Mycobacterium sp in modulating the innate immune response. We isolated amidated peptidoglycan from Mycobacterium sp and non-amidated peptidoglycan from Escherichia coli. We made a comparative analysis of the cytokine response produced on stimulation of innate immune cells by peptidoglycan from E. Coli and Mycobacterium sp. Macrophages and whole blood were treated with peptidoglycan and the cytokines secreted into spent medium and plasma respectively were analyzed using ELISA. Our results show that peptidoglycan from Mycobacterium sp is less effective in stimulating innate immune cells to produce cytokines. This intrinsic modulation of the cytokine response suggests that mycobacteria modify their peptidoglycan by amidation to evade innate immune response.
![Delegate Talk: Development of Supercritical Fluid Chromatography methods for the replacement of existing USP Normal phase liquid chromatography methods @ Amriteshwari Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/bioanalt.jpg)
Syed Salman Lateef and Vinayak A K
Development of Supercritical Fluid Chromatography methods for the replacement of existing USP Normal phase liquid chromatography methods
Normal phase liquid chromatography methods often have long run times and involve environmentally toxic/costly solvents. Supercritical chromatography methods on the other hand are faster, inexpensive, and eco-friendly. The low viscous supercritical carbon dioxide operates at high flow rates compared to LC without losing separation efficiency. In this work, SFC methods are developed to replace three United States Pharmacopeial (USP) normal phase achiral methods – prednisolone, tolazamide and cholecalciferol. System suitability parameters of the normal phase method are compared against the SFC method. Precision, linearity and robustness of the new SFC methods are demonstrated. SFC methods were found to be cost effective in terms of analysis time and solvent savings. The SFC method does not require purchase and disposal of expensive environmentally hazardous chemicals. Hence, the newly developed SFC method provides a faster and safer solution.
Sudarslal S, Ph.D.
Associate Professor, School of Biotechnology, Amrita University
Electrospray ionization ion trap mass spectrometry for cyclic peptide characterization
There has been considerable interest in the isolation and structural characterization of bioactive peptides produced by bacteria and fungi. Most of the peptides are cyclic depsipeptides characterized by the presence of lactone linkages and β-hydroxy fatty acids. Occurrence of microheterogeneity is another remarkable property of these peptides. Even if tandem mass spectrometers are good analytical tools to structurally characterize peptides and proteins, sequence analysis of cyclic peptides is often ambiguous due to the random ring opening of the peptides and subsequent generation of a set of linear precursor ions with the same m/z. Here we report combined use of chemical derivatization and multistage fragmentation capability of ion trap mass spectrometers to determine primary sequences of a series of closely related cyclic peptides.
![Delegate Talk: Bioanalytical Characterization of Therapeutic Proteins @ Amriteshwari Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/bioanalt.jpg)
Ravindra Gudihal, Suresh Babu C V
Bioanalytical Characterization of Therapeutic Proteins
The characterization of therapeutic proteins such as monoclonal antibody (mAb) during different stages of manufacturing is crucial for timely and successful product release. Regulatory agencies require a variety of analytical technologies for comprehensive and efficient protein analysis. Electrophoresis-based techniques and liquid chromatography (LC) either standalone or coupled to mass spectrometry (MS) are at the forefront for the in-depth analysis of protein purity, isoforms, stability, aggregation, posttranslational modifications, PEGylation, etc. In this presentation, a combination of various chromatographic and electrophoretic techniques such as liquid-phase isoelectric focusing, microfluidic and capillary-based electrophoresis (CE), liquid chromatography (LC) and combinations of those with mass spectrometry techniques will be discussed. We present a workflow based approach to the analysis of therapeutic proteins. In successive steps critical parameters like purity, accurate mass, aggregation, peptide sequence, glycopeptide and glycan analysis are analyzed. In brief, the workflow involved proteolytic digestion of therapeutic protein for peptide mapping, N-Glycanase and chemical labeling reaction for glycan analysis, liquid-phase isoelectric focusing for enrichment of charge variants followed by a very detailed analysis using state of the art methods such as CE-MS and LC-MS. For the analysis of glycans, we use combinations of CE-MS and LC-MS to highlight the sweet spots of these techniques. CE-MS is found to be more useful in analysis of highly sialylated glycans (charged glycans) while nano LC-MS seems to be better adapted for analysis of neutral glycans. These two techniques can be used to get complementary data to profile all the glycans present in a given protein. In addition, microfluidic electrophoresis was used as a QC tool in initial screening for product purity, analysis of papain digestion fragments of mAb, protein PEGylation products, etc. The described workflow involves multiple platforms, provides an end to end solution for comprehensive protein characterization and aims at reducing the total product development time.