Qualification: 
Ph.D
krishnakumarmenon@aims.amrita.edu

Dr. Krishnakumar Menon is an Associate Professor at Amrita Centre for Nanosciences and Molecular Medicine (ACNSMM) and currently heads the Clinical Proteomics and Translational Research Laboratory. Dr. Menon obtained M. Sc. in Biochemistry from Department of Biochemistry, M. S. University of Baroda, India and M. Sc.-Ph. D. on a scholarship equivalent to commonwealth research grant from National Multiple Sclerosis (MS) Society of Australia at Monash-La Trobe University, Australia. He is a recipient of student honor from La Trobe University and the prestigious JSPS fellowship from Japanese government to carry out oligosaccharide changes associated with myelin forming cells at the National Institute for Physiological Sciences, Japan. Subsequently, he was a fellow at the University of Connecticut Health Center, USA, as Research Investigator and Assistant Research Scientist at the University of Iowa hospitals, USA and the Monash Immunology and Stem Cell Laboratories at Monash University, Australia before joining as an Associate Professor at ACNSMM.  Dr. Menon’s main objective is to diagnose MS early and develop therapies for treating MS. In this respect, Dr. Menon developed different techniques for identifying novel biomarkers of diagnostic, prognostic and therapeutic significance in MS. His laboratory is supported by different grants from ICMR, DBT including Indo- Australian and Novartis global research grants. Currently, the lab’s focus is to take their research findings to the clinic for the benefit of patients.

Publications

Publication Type: Journal Article

Year of Publication Publication Type Title

2018

Journal Article

S. Chandrashekaran, Karthikeyan, S., Balakrishnan, A., Nair, S., Kumar, M. Kumaran Sa, Vattathara, J. Jose, and Krishnakumar N. Menon, “Expression and Purification of Quinine Dihydro Pteridine Reductase from astrocytes and its significance in the astrocyte pathology.”, Int J Biol Macromol, 2018.[Abstract]


Quinine dihydropteridinereductase (QDPR) is involved in the synthesis of tetradihydrobiopteridine (BH4) that serve as cofactor for many aromatic hydroxylases including induced nitric oxide synthase (NOS) leading to NO production. Increased activity of QDPR has been associated with decrease levels of TGF-β, a cytokine that regulates the immune response and that elevated levels of NO has been associated with neurodegenerative diseases. Thus, expression of QDPR in astrocytes is essential to study the pathological changes observed in many neurodegenerative disorders. We have expressed QDPR in astrocytes and generated stably expressing clones that overexpresses QDPR. We further verified the specificity of QDPR expression using immunofluorescence and immunoblotting. To further confirm, we purified QDPR using Ni-NTA column and subjected the purified fraction to immunoblotting using anti-QDPR antibody and identified two major protein products of QDPR resolving at 25 and 17 kDa as reported in the literature. In order to further assess the significance of QDPR expression, we verified the expression of iNOS in QDPR over expressing cells. We show for the first time statistically significant up regulation of iNOS in QDPR overexpressing astrocytes. Increased expression of iNOS associated with astrocyte pathology seen in many neurodegenerative disorders may have implications in autoimmune neurodegenerative disorders.

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2018

Journal Article

V. S. Pillai, Kundargi, R. R., Edathadathil, F., Nair, S., Thilak, J., Mathew, R. A., Xavier, T., Shenoy, P., and Krishnakumar N. Menon, “Identification of Prolargin Expression in Articular Cartilage and its Significance in Rheumatoid Arthritis Pathology”, International Journal of Biological Macromolecules, 2018.[Abstract]


Qualitative 2D gel-electrophoresis (2DE) protein profiling for osteoarthritis (OA) and rheumatoid arthritis (RA) is challenging because of selective protein loss due to discrepancies in protein precipitation methodologies. Thus, we aimed at developing qualitative proteinrepresentation from OA/RA articular cartilage without protein precipitation towards identification of clinically relevant proteins. Chondroitinase digested human articular cartilages from RA patients were subjected to protein extraction using guanidinium hydrochloride (GuHCl) or 8 M urea with 10 or 2% ASB-14-4 or 0.45 M urea with 2% ASB-14-4 with cetylpyridinium chloride (CPC). The GuHCl extract is further protein precipitated with acetone or ammonium acetate-methanol or centricon-fractionated using 100 kDa cut filters and protein precipitated using ethanol. Processed extracts were subjected to 2DE to identify protein profiles. Poor proteins representations were observed in 2D gels with protein precipitated samples compared to qualitative protein representations seen in 2D gels of 0.45 M urea and 2%ASB-14-4 extraction procedure reproducibly. The strategy circumventing protein precipitation generated qualitative 2D gels. RA vs OA gel comparison showed elevated prolargin levels in RA with biglycan levels remaining unaltered. Up regulation of prolargin in RA suggests the likelihood of an adaptive mechanism to control the increased osteoclastogenesis in RA and may have therapeutic value in controlling the disease.

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2017

Journal Article

M. Kumaran Sa Kumar, Nair, S., Dr. Ullas Mony, Kalingavarman, S., Venkat, R., Sivanarayanan, T. Balakrishn, Unni, A. Kodakara K., Rajeshkannan, R., Anandakuttan, A., Radhakrishnan, S., and Krishnakumar N. Menon, “Significance of Elevated Prohibitin 1 levels in Multiple Sclerosis Patients Lymphocytes Towards the Assessment of Subclinical Disease Activity and its Role in the Central Nervous System Pathology of Disease.”, International Journal of Biological Macromolecules, 2017.[Abstract]


Multiple Sclerosis (MS) is an autoimmune-neurodegenerative disorder managed therapeutically by modulating lymphocytes activity which has potential in disease management. Prohibitin 1(PHB) that controls the reactive oxygen species (ROS) and present on the activated lymphocytes have significance in the therapy of MS as esters of fumaric acid that regulates ROS is in phase II/III clinical trials. Thus, we evaluated the expression levels of PHB1 in experimental autoimmune encephalomyelitis (EAE), the animal model of MS and on MS patient's lymphocytes. PHB levels in brain tissue of EAE animals were determined by immunoblotting and on blood lymphocytes from MS relapse, Remission, Optic Neuritis, Neurological controls and Healthy volunteers by FACS using anti-PHB and anti-CD45 antibodies. We observed significant elevation of PHB in EAE brains (91.0 ± 17.59%) vs controls (29.8 ± 12.9%) (p = 0.01) and on lymphocytes of MS patients in acute (73.5 ± 11.20%) or relapsing (69.3 ± 17.33%) phase compared to remission (45.9 ± 8.08%) [p = 0.034 acute vs remission; p = 0.004 relapse vs remission]. Up regulation of PHB in relapsing vs remission MS patients imply the potential use of PHB to clinically evaluate subclinical disease status towards prognosis of an oncoming relapse. Elevated PHB levels in EAE brains signify the role of PHB in regulating ROS and implies PHB's role in oxidative stress.

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2016

Journal Article

P. Sadanandan, Payne, N. L., Sun, G., Anusha Ashokan, Xavier, W., Gowd, S. G., Shantikumar V. Nair, Krishnakumar N. Menon, Bernard, C. C. A., and Koyakutty, M., “Nanoengineered Myelin Oligodendrocyte Glycoprotein Peptides Suppressed Experimental Autoimmune Encephalomyelitis: Implications In Multiple Sclerosis Therapy”, European Journal of Immunology, vol. 46 , 1 vol., pp. 316-317, 2016.

2015

Journal Article

M. G. Sangeet Nair, Dr. Ullas Mony, Dr. Deepthy Menon, Dr. Manzoor K., Sidharthan, N., Pavithran, K., Shantikumar V Nair, and Krishnakumar N. Menon, “Development and molecular characterization of polymeric micro-nanofibrous scaffold of a defined 3-D niche for in vitro chemosensitivity analysis against acute myeloid leukemia cells”, International journal of nanomedicine, 2015.[Abstract]


Standard in vitro drug testing employs 2-D tissue culture plate systems to test anti-leukemic drugs against cell adhesion-mediated drug-resistant leukemic cells that harbor in 3-D bone marrow microenvironments. This drawback necessitates the fabrication of 3-D scaffolds that have cell adhesion-mediated drug-resistant properties similar to in vivo niches. We therefore aimed at exploiting the known property of polyurethane (PU)/poly-L-lactic acid (PLLA) in forming a micro-nanofibrous structure to fabricate unique, not presented before, as far as we are aware, 3-D micro-nanofibrous scaffold composites using a thermally induced phase separation technique. Among the different combinations of PU/PLLA composites generated, the unique PU/PLLA 60:40 composite displayed micro-nanofibrous morphology similar to decellularized bone marrow with increased protein and fibronectin adsorption. Culturing of acute myeloid leukemia (AML) KG1a cells in FN-coated PU/PLLA 60:40 shows increased cell adhesion and cell adhesion-mediated drug resistance to the drugs cytarabine and daunorubicin without changing the original CD34(+)/CD38(-)/CD33(-) phenotype for 168 hours compared to fibronectin tissue culture plate systems. Molecularly, as seen in vivo, increased chemoresistance is associated with the upregulation of anti-apoptotic Bcl2 and the cell cycle regulatory protein p27(Kip1) leading to cell growth arrest. Abrogation of Bcl2 activity by the Bcl2-specific inhibitor ABT 737 led to cell death in the presence of both cytarabine and daunorubicin, demonstrating that the cell adhesion-mediated drug resistance induced by Bcl2 and p27(Kip1) in the scaffold was similar to that seen in vivo. These results thus show the utility of a platform technology, wherein drug testing can be performed before administering to patients without the necessity for stromal cells.

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2013

Journal Article

Krishnakumar N. Menon, Haridasan, A., Xavier, T., Kundargi, R., and Nair, S., “Neuroproteomics: Are We Biased in Our Representation of Molecular Targets Associated with Specific Domains? Implications in Biomarker Discovery”, Current Proteomics, vol. 10, pp. 56-66, 2013.[Abstract]


The central nervous system (CNS) encompasses complex groups of cells that coexist to form the functional units of brain mainly comprising three cell types: neurons, oligodendrocytes and astrocytes. The network of these cells through interaction between each other leads to formation of complex organizations such as nodes of ranvier, paranodes, juxtaparanodes etc. Therefore, regions of molecular complexity in brain have different protein/lipid combinatorial complexities. Isolation and enrichment of these domains/regions are crucial for the maximum representation of the protein complement,which otherwise goes undetected due to its miniscule amounts in the whole brain homogenate. Thus preparation of enriched fractions representing the proteins of these complexities is important for detection of molecules of pathogenic and diagnostic significance in diseases. Apart from enrichment, the proteins from the enriched fractions have to be solubilized into an appropriate sample format suitable for resolving into its individual protein components. This holds the key to identification of novel molecules important in neurodegenerative diseases. Thus, sample processing for understanding the organizational and functional proteomics becomes an extremely important step for fruitful results to avoid neuroproteomics biases. This review focuses on the functional units of brain, significance of certain molecules in these organizations and how current techniques are meaningfully employed towards neuroproteomics. In addition, we review how custom techniques of sample preparation for protein analysis are being tailored to address issues for a better analysis of protein complement from specialized regions of the functional units of brain in discovering biomarkers pertaining to neurodegenerative diseases. © 2013 Bentham Science Publishers.

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2011

Journal Article

S. Nair, Xavier, T., Kumar, M. K. Satheesh, Saha, S., and Krishnakumar N. Menon, “Exploitation of Detergent Thermodynamics in the Direct Solubilization of Myelin Membrane Proteins for Two-dimensional Gel Electrophoresis for Proteomic Analysis”, Electrophoresis, vol. 32, pp. 3621-3629, 2011.[Abstract]


Performing 2-DE of lipid-rich multilamellar membranes like myelin is a cumbersome task. However, for understanding its molecular organization and changes during diseases, identification of proteins of myelin is essential. Although the 2-D-proteomic approach of myelin has been employed to understand the myelin proteome, representation of myelin proteins in its entirety is still a challenge. 2-DE profiling of myelin proteins is very important for the detection of immuno-reactivity to myelin proteins from various biological fluids following Western blotting in diseases like multiple sclerosis. Here we developed a novel approach by exploiting the thermodynamic principles behind detergent-mediated solubilization of myelin membranes without any conventional processing of myelin involving precipitation of myelin proteins. We show that the addition of myelin to ASB-14-4 resulted in significant increase in protein representation of myelin in 2-DE compared with the addition of ASB-14-4 to myelin. Moreover, the number and resolution of spots are significantly higher in myelin to ASB-14-4 strategy than other strategies of myelin sample processing such as ASB-14-4 to myelin or ethanol or acetone or methanol-ammonium acetate precipitation of myelin proteins. In addition, the step involves no precipitation that selective removal of any proteins as a result of precipitation is nil and a qualitative representation of myelin proteins in a 2-D gel is achieved.

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2011

Journal Article

Krishnakumar N. Menon, Steer, D. L., Short, M., Petratos, S., Smith, I., and Bernard, C. C. A., “A Novel Unbiased Proteomic Approach to Detect the Reactivity of Cerebrospinal Fluid in Neurological Diseases”, Molecular and Cellular Proteomics, vol. 10, 2011.[Abstract]


Neurodegenerative diseases, such as multiple sclerosis represent global health issues. Accordingly, there is an urgent need to understand the pathogenesis of this and other central nervous system disorders, so that more effective therapeutics can be developed. Cerebrospinal fluid is a potential source of important reporter molecules released from various cell types as a result of central nervous system pathology. Here, we report the development of an unbiased approach for the detection of reactive cerebrospinal fluid molecules and target brain proteins from patients with multiple sclerosis. To help identify molecules that may serve as clinical biomarkers for multiple sclerosis, we have biotinylated proteins present in the cerebrospinal fluid and tested their reactivity against brain homogenate as well as myelin and myelin-axolemmal complexes. Proteins were separated by two-dimensional gel electrophoresis, blotted onto membranes and probed separately with biotinylated unprocessed cerebrospinal fluid samples. Protein spots that reacted to two or more multiple sclerosis-cerebrospinal fluids 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 multiple sclerosis cerebrospinal fluid, such as αβ crystallin, enolase, and 14-3-3-protein, we have identified several additional molecules involved in mitochondrial and energy metabolism, myelin gene expression and/or cytoskeletal organization. These include aspartate aminotransferase, cyclophilin-A, quaking protein, collapsin response mediator protein-2, ubiquitin carboxy-terminal hydrolase L1, and cofilin. To further validate these findings, 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.

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2010

Journal Article

T. Xavier, ,, Ganesan, T. S., and Krishnakumar N. Menon, “A Simple and Efficient Method for Processing of Cell Lysates for Two-dimensional Gel Electrophoresis”, Electrophoresis, vol. 31, pp. 2429-2435, 2010.[Abstract]


Sample preparation is one of the major issues in 2-DE for the separation of proteins. Although a 100% representation of cellular proteins onto a 2-DE is virtually impossible, maximum representation of cellular proteins compared with the original cell lysate is important in the subsequent analysis. We demonstrate that lysis of cells in urea/thiourea solution with subsequent sonication to disrupt the nucleic acids and concentration of the lysate using centri-con led to enrichment of proteins. The procedure resulted in minimal nucleic acid contamination with better resolution of spots. 2-DE spot patterns of proteins prepared using urea - thiourea solubilization/centri-con method to other protein enrichment methods such as phenol/chloroform/isoamyl alcohol extraction, methanol/ammonium acetate precipitation, acetone precipitation and ethanol precipitation were compared. Urea - thiourea solubilization combined with centri-con method of protein enrichment represented higher number/unique spots particularly in the 50-250 kDa M r compared with others. Lysis of cells in urea/thiourea from the beginning of lysate preparation preserves the proteins from protease activity due to denaturation of proteases. Thus, we demonstrate that the centri-con methodology is simple and effective for the preparation of high-quality sample that can be used for a qualitative representation of cellular proteins on a 2-DE for proteomic analysis.

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