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.
Lalitha Subramanian, Ph.D.
Chief Scientific Officer & VP, Services at Scienomics, USA
Nanoscale Simulations – Tackling Form and Formulation Challenges in Drug Development and Drug Delivery
Lalitha Subramanian, Dora Spyriouni, Andreas Bick, Sabine Schweizer, and Xenophon Krokidis Scienomics
The discovery of a compound which is potent in activity against a target is a major milestone in Pharmaceutical and Biotech industry. However, a potent compound is only effective as a therapeutic agent when it can be administered such that the optimal quantity is transported to the site of action at an optimal rate. The active pharmaceutical ingredient (API) has to be tested for its physicochemical properties before the appropriate dosage form and formulation can be designed. Some of the commonly evaluated parameters are crystal forms and polymorphs, solubility, dissolution behavior, stability, partition coefficient, water sorption behavior, surface properties, particle size and shape, etc. Pharmaceutical development teams face the challenge of quickly and efficiently determining a number of properties with small quantities of the expensive candidate compounds. Recently the trend has been to screen these properties as early as possible and often the candidate compounds are not available in sufficient quantities. Increasingly, these teams are leveraging nanoscale simulations similar to those employed by drug discovery teams for several decades. Nanoscale simulations are used to predict the behavior using very little experimental data and only if this is promising further experiments are done. Another aspect where nanoscale simulations are being used in drug development and drug delivery is to get insights into the behavior of the system so that process failures can be remediated and formulation performance can be improved. Thus, the predictive screening and the in-depth understanding leads to experimental efficiency resulting in far-reaching business impacts.
With specific examples, this talk will focus on the different types of nanoscale simulations used to predict properties of the API in excipients and also provide insight into system behavior as a function of shelf life, temperature, mechanical stress, etc.
Manjunath Joshi, Apoorva Lad, Bharat Prasad Alevoor, Aswath Balakrishnan, Lingadakai Ramachandra and Kapaettu Satyamoorthy
Pathological conditions during Type 2 Diabetes (T2D) are associated with elevated risk for common community acquired infections due to poor glycemic control. Multiple studies have indicated specific defects in innate and adaptive immune function in diabetic subjects. Neutrophils play an important role in eliminating pathogens as an active constituent of innate immune system. Apart from canonically known phagocytosis mechanism, neutrophils are endowed with a unique ability to produce extracellular traps (NETs) to kill pathogens by expelling DNA coated with bactericidal proteins and histone. NETosis is stimulated by diverse bacteria and their products, fungi, protozoans, cytokines, phorbol esters and by activated platelets. Considering deregulation of metabolic and immune response pathways during pathological state of diabetes and NETosis as a potential mechanism for killing bacteria, we therefore, investigated whether hyperglycemic conditions modulate formation of neutrophil NETs and attempted to identify underlying immunoregulatory mechanisms. Freshly isolated neutrophils from normal individuals were cultured in absence or presence of high glucose (different concentrations) for 24 hours and activated with either LPS (2 mg/ml) or PMA (20 ng/ml) or IL-6 (20 ng/ml) for 3 hours. NETs were visualized and quantified by addition of DNA binding dye SYTOX green using fluorescence microscope and fluorimetry. NETs were quantified in Normal and diabetic subjects. Serum IL-6 levels were measured using ELISA technique. NETs bound elasatse were quantified in normal and diabetic subjects in presence or absence of DNase. Bacterial killing assays were performed upon infecting E.coli with activated neutrophils from normal and diabetic subjects. Microscopy and fluorimetry analysis suggested dramatic impairment in NETs formation under high glucose conditions. Extracellular DNA lattices formed in hyperglycemic conditions were short lived and unstable leading to rapid disintegration. Subsequent, time course experiments showed that NETs production was delayed in hyperglycemic conditions. To validate our findings more closely to clinical conditions, we investigated the neutrophil activation and NETs formation in diabetic patients. Upon stimulation with LPS for three hours, neutrophils from diabetic subjects responded weakly to LPS and lesser NETs were formed; whereas, neutrophils from normal individuals showed robust release of NETs. In few patients we found short and imperfect NETs in basal conditions suggesting constitutive activation of neutrophils in diabetic subjects. Interestingly, NETs bound elastase activity was reduced in diabetes subjects when compared to non-diabetic individuals, indicating a dysfunction of one of the important protein component of NETs during diabetes. Neutrophils from diabetic subjects released higher levels of IL-6 without any stimulation suggesting an existence of constitutively activated pro-inflammatory state. IL-6 induced NETs formation and was abrogated by high glucose. Weobserved that glycolysis inhibitor 2-DG resensitize the high glucose attenuated LPS and IL-6 induced NETs. a) NETs are influenced by glucose homeostasis, b) IL-6 as potent inducer of energy dependent NETs formation and c) hyperglycemia mimics a state of constitutively active pro-inflammatory condition in neutrophils leading to reduced response to external stimuli making diabetic subjects susceptible for infections.
Jeff Perry, Ph.D.
Assistant Professor, University of California, Riverside
Combined Crystallography and SAXS Methods for Studying Macromolecular Complexes
Recent developments in small angle X-ray scattering (SAXS) are rapidly providing new insights into protein interactions, complexes and conformational states in solution, allowing for detailed biophysical quantification of samples of interest1. Initial analyses provide a judgment of sample quality, revealing the potential presence of aggregation, the overall extent of folding or disorder, the radius of gyration, maximum particle dimensions and oligomerization state. Structural characterizations may include ab initio approaches from SAXS data alone, or enhance structural solutions when combined with previously determined crystal/NMR domains. This combination can provide definitions of architectures, spatial organizations of the protein domains within a complex, including those not yet determined by crystallography or NMR, as well as defining key conformational states. Advantageously, SAXS is not generally constrained by macromolecule size, and rapid collection of data in a 96-well plate format provides methods to screen sample conditions. Such screens include co-factors, substrates, differing protein or nucleotide partners or small molecule inhibitors, to more fully characterize the variations within assembly states and key conformational changes. These analyses are also useful for screening constructs and conditions that are most likely to promote crystal growth. Moreover, these high throughput structural determinations can be leveraged to define how polymorphisms affect assembly formations and activities. Also, SAXS-based technologies may be potentially used for novel structure-based screening, for compounds inducing shape changes or associations/diassociations. This is addition to defining architectural characterizations of complexes and interactions for systems biology-based research, and distinctions in assemblies and interactions in comparative genomics. Thus, SAXS combined with crystallography/NMR and computation provides a unique set of tools that should be considered as being part of one’s repertoire of biophysical analyses, when conducting characterizations of protein and other macromolecular interactions.
1 Perry JJ & Tainer JA. Developing advanced X-ray scattering methods combined with crystallography and computation. Methods. 2013 Mar;59(3):363-71.
