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.
K. P. Mohanakumar, Ph.D.
Chief Scientist, Cell Biology & Physiology Division, Indian Institute of Chemical Biology, Kolkata
Neuroprotective and neurodestructive effects of Ayurvedic drug constituents: Parkinson’s disease
The present study reports the good and the bad entities in an Indian traditional medicine used for treating Parkinson’s disease (PD). A prospective clinical trial on the effectiveness of Ayurvedic medication in a population of PD patients revealed significant benefits, which has been attributed to L-DOPA present in the herbs [1]. Later studies revealed better benefits with one of the herbs alone, compared to pure L-DOPA in a clinical trial conducted in UK [2], and in several studies conducted on animal models of PD in independent laboratories world over [3-5]. We have adapted strategies to segregate molecules from the herb, and then carefully removed L-DOPA contained therein, and tested each of these sub-fractions for anti-PD activity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, rotenone and 6-hydroxydopamine -induced parkinsonian animal models, and transgenic mitochondrial cybrids. We report here two classes of molecules contained in the herb, one of which possessed severe pro-parkinsonian (phenolic amine derivatives) and the other having excellent anti-parkinsonian potential (substituted tetrahydroisoquinoline derivatives). The former has been shown to cause severe dopamine depletion in the striatum of rodents, when administered acutely or chronically. It also caused significant behavioral aberrations, leading to anxiety and depression [6]. The latter class of molecules administered in PD animal model [7], caused reversal of behavioral dysfunctions and significant attenuation of striatal dopamine loss. These effects were comparable or better than the effects of the anti-PD drugs, selegiline or L-DOPA. The mechanism of action of the molecule has been found to be novel, at the postsynaptic receptor signaling level, as well as cellular α-synuclein oligomerization and specifically at mitochondria. The molecule helped in maintaining mitochondrial ETC complex activity and stabilized cellular respiration, and mitochondrial fusion-fission machinery with specific effect on the dynamin related protein 1. Although there existed significant medical benefits that could be derived to patients due to the synergistic actions of several molecules present in a traditional preparation, accumulated data in our hands suggest complicated mechanisms of actions of Ayurvedic medication. Our results also provide great hope for extracting, synthesizing and optimizing the activity of anti-parkinsonian molecules present in traditional Ayurvedic herbs, and for designing novel drugs with novel mechanisms of action.
- N, Nagashayana, P Sankarankutty, MRV Nampoothiri, PK Mohan and KP Mohanakumar, J Neurol Sci. 176, 124-7, 2000.
- Katzenschlager R, Evans A, Manson A, Patsalos PN, Ratnaraj N, Watt H, Timmermann L, Van der Giessen R, Lees AJ. J Neurol Neurosurg Psychiatry.75, 1672-7, 2004.
- Manyam BV, Dhanasekaran M, Hare TA. Phytother Res. 18, 706-12, 2004.
- Kasture S, Pontis S, Pinna A, Schintu N, Spina L, Longoni R, Simola N, Ballero M, Morelli M. Neurotox Res. 15, 111-22, 2009.
- Lieu CA, Kunselman AR, Manyam BV, Venkiteswaran K, Subramanian T. Parkinsonism Relat Disord.16, 458-65, 2010.
- T Sengupta and KP Mohanakumar, Neurochem Int. 57, 637-46, 2010.
- T Sengupta, J Vinayagam, N Nagashayana, B Gowda, P Jaisankar and KP Mohanakumar, Neurochem Res 36, 177-86, 2011
Nader Pourmand, Ph.D.
Director, UCSC Genome Technology Center,University of California, Santa Cruz
Biosensor and Single Cell Manipulation using Nanopipettes
Approaching sub-cellular biological problems from an engineering perspective begs for the incorporation of electronic readouts. With their high sensitivity and low invasiveness, nanotechnology-based tools hold great promise for biochemical sensing and single-cell manipulation. During my talk I will discuss the incorporation of electrical measurements into nanopipette technology and present results showing the rapid and reversible response of these subcellular sensors to different analytes such as antigens, ions and carbohydrates. In addition, I will present the development of a single-cell manipulation platform that uses a nanopipette in a scanning ion-conductive microscopy technique. We use this newly developed technology to position the nanopipette with nanoscale precision, and to inject and/or aspirate a minute amount of material to and from individual cells or organelle without comprising cell viability. Furthermore, if time permits, I will show our strategy for a new, single-cell DNA/ RNA sequencing technology that will potentially use nanopipette technology to analyze the minute amount of aspirated cellular material.
Karmeshu, Ph.D.
Dean & Professor, School of Computer & Systems Sciences & School of Computational & Integrative Sciences, Jawaharlal Nehru University, India.
Interspike Interval Distribution of Neuronal Model with distributed delay: Emergence of unimodal, bimodal and Power law
The study of interspike interval distribution of spiking neurons is a key issue in the field of computational neuroscience. A wide range of spiking patterns display unimodal, bimodal ISI patterns including power law behavior. A challenging problem is to understand the biophysical mechanism which can generate the empirically observed patterns. A neuronal model with distributed delay (NMDD) is proposed and is formulated as an integro-stochastic differential equation which corresponds to a non-markovian process. The widely studied IF and LIF models become special cases of this model. The NMDD brings out some interesting features when excitatory rates are close to inhibitory rates rendering the drift close to zero. It is interesting that NMDD model with gamma type memory kernel can also account for bimodal ISI pattern. The mean delay of the memory kernels plays a significant role in bringing out the transition from unimodal to bimodal ISI distribution. It is interesting to note that when a collection of neurons group together and fire together, the ISI distribution exhibits power law.
Tim Guilliams, Ph.D.
Junior Associate Fellow at the Centre for Science and Policy, University of Cambridge
From Camels to Worms: Novel Approaches for Drug Discovery in Parkinson’s Disease
The discovery of novel treatments for neurodegenerative diseases, such as Parkinson’s disease, represents one of the biggest scientific challenges of the 21st century. The development of new tools and models to study the mechanisms underlying neurotoxicity is therefore essential. During my talk, I will outline new strategies for drug design and innovation used during my PhD at the University of Cambridge, which include the combination of fluorescent nematode worms, camelid antibody fragment technology and chemical compounds. These novel approaches will help us to gain insights into the key pathogenic steps involved in Parkinson’s disease and potentially lead to new therapeutic strategies.
![Delegate Talk: Inflammation Induced Epigenetic Changes in Endothelial Cells: Role in Vascular Insulin Resistance @ Acharya Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/gtm.jpg)
Aswath Balakrishnan, Kapaettu Satyamoorthy and Manjunath B Joshi
Introduction
Insulin resistance is a hall mark of metabolic disorders such as diabetes. Reduced insulin response in vasculature leads to disruption of IR/Akt/eNOS signaling pathway resulting in vasoconstriction and subsequently to cardiovascular diseases. Recent studies have demonstrated that inflammatory regulator interleukin-6 (IL-6), as one of the potential mediators that can link chronic inflammation with insulin resistance. Accumulating evidences suggest a significant role of epigenetic mechanisms such as DNA methylation in progression of metabolic disorders. Hence the present study aimed to understand the role of epigenetic mechanisms involved during IL-6 induced vascular insulin resistance and its consequences in cardiovascular diseases.
Materials and Methods
Human umbilical vein endothelial cells (HUVEC) and Human dermal microvascular endothelial cells (HDMEC) were used for this study. Endothelial cells were treated in presence or absence of IL-6 (20ng/ml) for 36 hours and followed by insulin (100nM) stimulation for 15 minutes. Using immunoblotting, cell lysates were stained for phosphor- and total Akt levels to measure insulin resistance. To investigate changes in DNA methylation, cells were treated with or without neutrophil conditioned medium (NCM) as a physiological source of inflammation or IL-6 (at various concentrations) for 36 hours. Genomic DNA was processed for HPLC analysis for methyl cytosine content and cell lysates were analyzed for DNMT1 (DNA (cytosine-5)-methyltransferase 1) and DNMT3A (DNA (cytosine-5)-methyltransferase 3A) levels using immunoblotting.
Results
Endothelial cells stimulated with insulin exhibited an increase in phosphorylation of Aktser 473 in serum free conditions but such insulin response was not observed in cells treated with IL-6, suggesting chronic exposure of endothelial cells to IL-6 leads to insulin resistance. HPLC analysis for global DNA methylation resulted in decreased levels of 5-methyl cytosine in cells treated with pro-inflammatory molecules (both by NCM and IL-6) as compared to untreated controls. Subsequently, analysis in cells treated with IL-6 showed a significant decrease in DNMT1 levels but not in DNMT3A. Other pro-inflammatory marker such as TNF-α did not exhibit such changes.
Conclusion
Our study suggests: a) Chronic treatment of endothelial cells with IL-6 results in insulin resistance b) Neutrophil conditioned medium and IL-6 decreases methyl cytosine levels c) DNMT1 but not DNMT3a levels are reduced in cells treated with IL-6.
Akhilesh Pandey, Ph.D.
Professor, Johns Hopkins University School of Medicine, Baltimore, USA
A draft map of the human proteome
We have generated a draft map of the human proteome through a systematic and comprehensive analysis of normal human adult tissues, fetal tissues and hematopoietic cells as an India-US initiative. This unique dataset was generated from 30 histologically normal adult tissues, fetal tissues and purified primary hematopoietic cells that were analyzed at high resolution in the MS mode and by HCD fragmentation in the MS/MS mode on LTQ-Orbitrap Velos/Elite mass spectrometers. This dataset was searched against a 6-frame translation of the human genome and RNA-Seq transcripts in addition to standard protein databases. In addition to confirming a large majority (>16,000) of the annotated protein-coding genes in humans, we obtained novel information at multiple levels: novel protein-coding genes, unannotated exons, novel splice sites, proof of translation of pseudogenes (i.e. genes incorrectly annotated as pseudogenes), fused genes, SNPs encoded in proteins and novel N-termini to name a few. Many proteins identified in this study were identified by proteomic methods for the first time (e.g. hypothetical proteins or proteins annotated based solely on their chromosomal location). We have generated a catalog of proteins that show a more tissue-restricted pattern of expression, which should serve as the basis for pursuing biomarkers for diseases pertaining to specific organs. This study also provides one of the largest sets of proteotypic peptides for use in developing MRM assays for human proteins. Identification of several novel protein-coding regions in the human genome underscores the importance of systematic characterization of the human proteome and accurate annotation of protein-coding genes. This comprehensive dataset will complement other global HUPO initiatives using antibody-based as well as MRM mass spectrometry-based strategies. Finally, we believe that this dataset will become a reference set for use as a spectral library as well as for interesting interrogations pertaining to biomedical as well as bioinformatics questions.