Krishnakumar Menon, Ph.D.
Associate Professor, Centre for Nanosciences & Molecular Medicine, Amrita University, Kochi, India
A Far-Western Clinical Proteomics Approach to Detect Molecules of Clinical and Pathological Significance in the Neurodegenerative Disease Multiple Sclerosis.
Multiple Sclerosis (MS), an autoimmune neurodegenerative disorder of the central nervous system. The disease affects young adults at their prime age leading to severe debilitation over several years. Despite advances in MS research, the cause of the disease remains elusive. Thus, our objective is to identify novel molecules of pathological and diagnostic significance important in the understanding, early diagnosis and treatment of MS. Biological fluids such as cerebrospinal fluid (CSF), that bathe the brain serve as a potential source for the identification of pathologically significant autoantibody reactivity in MS. In this regard, we report the development of an unbiased clinical proteomics approach for the detection of reactive CSF molecules that target brain proteins from patients with MS. Proteins of myelin and myelin-axolemmal complexes were separated by two-dimensional gel electrophoresis, blotted onto membranes and probed separately with biotinylated unprocessed CSF samples. Protein spots that reacted specifically to MS-CSF’s were further analyzed by matrix assisted laser desorption ionization-time-of-flight time-of-flight mass spectrometry. In addition to previously reported proteins found in MS, we have identified several additional molecules involved in mitochondrial and energy metabolism, myelin gene expression and/or cytoskeletal organization. Among these identified molecules, the cellular expression pattern of collapsin response mediator protein-2 and ubiquitin carboxy-terminal hydrolase L1 were investigated in human chronic-active MS lesions by immunohistochemistry. The observation that in multiple sclerosis lesions phosphorylated collapsin response mediator protein-2 was increased, whereas Ubiquitin carboxy-terminal hydrolase L1 was down-regulated, not only highlights the importance of these molecules in the pathology of this disease, but also illustrates the use of our approach in attempting to decipher the complex pathological processes leading to multiple sclerosis and other neurodegenerative diseases. Further, we show that in clinicaly isolated syndrome (CIS), we could identify important molecules that could serve as an early diagnostic biomarker in MS.
Claudia AM Wheeler-Kingshott, Ph.D.
University Reader in Magnetic Resonance Physics, Department of Neuroinflammation, UCL Institute of Neurology, London, UK
Abstract
Detecting neuronal activity in vivo non-invasively is possible with a number of techniques. Amongst these, in 1990 functional magnetic resonance imaging (fMRI) was proposed as a technique that has a great ability to spatially map brain activity by exploiting the blood oxygenation level dependent (BOLD) contrast mechanism [1, 2]. In fact, neuronal activation triggers a demand for oxygen and induces a localised increase in blood flow and blood volume, which actually exceeds the metabolic needs. This in turns causes an increase of oxyhaemoglobin in the venous compartment, which is a transient phenomenon and is accompanied by a transient change (decrease) in the concentration of deoxyhaemoglobin. Due to its paramagnetic properties, the amount of deoxyhaemoglobin present in the venous blood affects the local magnetic field seen by the spins (protons) and determines the local properties of the MR signal. A decrease in deoxyhaemoglobin during neuronal activity, therefore, induces local variations of this magnetic field that increases the average transverse relaxation time of tissue, measured via the T2* parameter [3]. This means that there is an increase of the MR signal (of the order of a few %, typically <5%) linked to metabolic changes happening during brain function. Activation can be inferred at different brain locations by performing tasks while acquiring the MR signal and comparing periods of rest to periods of activity.
The macroscopic changes of the BOLD signal are well characterised, while the reason for the increased blood supply, exceeding demands, needs further thoughts. Here we will discuss two approaches for explaining the BOLD phenomenon, one that links it to adenosine triphosphate production [4] and enzyme saturation, the other that relates it to the very slow diffusion of oxygen through the blood-brain-barrier with a consequent compensatory high demand of oxygen [5]. Some evidence of restricted oxygen diffusion has been shown by means of hypercapnia [6], although it is not excluded that both mechanisms may be present.
Overall, the BOLD signal changes theory and its physiological basis will be presented and discussed.
References
- Ogawa, S., et al., Brain magnetic resonance imaging with contrast dependent on blood oxygenation. Proc Natl Acad Sci U S A, 1990. 87(24): p. 9868-72.
- Kwong, K.K., et al., Dynamic magnetic resonance imaging of human brain activity during primary sensory stimulation. Proc Natl Acad Sci U S A, 1992. 89(12): p. 5675-9.
- Bandettini PA, et al. Spin-echo and gradient-echo EPI of human brain activation using BOLD contrast: a comparative study at 1.5 T. NMR Biomed. 1994 Mar;7(1-2):12-20
- Fox, P.T., et al., Nonoxidative glucose consumption during focal physiologic neural activity. Science, 1988. 241(4864): p. 462-4.
- Gjedde, A., et al. Reduction of functional capillary density in human brain after stroke. J Cereb Blood Flow Metab, 1990. 10(3): p. 317-26.
- Hoge, R.D., et al., Linear coupling between cerebral blood flow and oxygen consumption in activated human cortex. Proc Natl Acad Sci U S A, 1999. 96(16): p. 9403-8.
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.
Kal Ramnarayan, Ph.D.
Co-founder President & Chief Scientific Officer, Sapient Discovery, San Diego, CA, USA
A cost-effective approach to Protein Structure-guided Drug Discovery: Aided by Bioinformatics, Chemoinformatics and computational chemistry
With the mapping of the human genome completed almost a decade ago, efforts are still underway to understand the gene products (i.e., proteins) in the human biological and disease pathways. Deciphering such information is very important for the discovery and development of small molecule drugs as well as protein therapeutics for various human diseases for which no cure exists. As an example, with more than 500 members, the kinase family of protein targets continues to be an important and attractive class for drug discovery. While how many of the members in this family are actually druggable is still to be established, there are several ongoing efforts on this class of proteins across a broad spectrum of disease categories. Even though in general the protein structural topology might looks similar, there are issues with respect selectivity of identified small molecule inhibitors when, the lead molecule discovery is carried out at the ATP binding site. As an added complexity, allosteric modulators are needed for some of the members, but the actual site for such modulation on the protein target can not resolved with uncertainty. In this presentation we will describe a bioinformatics and computational based platform for small molecule discovery for protein targets that are involved in protein-protein interactions as well as targets like kinases and phosphatases. We will describe a computational approach in which we have used an informatics based platform with several hundred kinases to sort through in silico and identify inhibitors that are likely to be highly selective in the lead generation phase. We will discuss the implication of this approach on the drug discovery of the kinase and phosphatase classes in general and independent of the disease category.