Centre for Nanosciences
Amrita Institute of Medical Sciences,
Amrita Vishwa Vidyapeetham
AIMS Ponekkara P. O., Kochi, Kerala - 682 041, India.

0484 285 8750
researchsecretary@aims.amrita.edu
 

 

Dr. Krishnakumar Menon is an associate professor at 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 Asst 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.

The laboratory of Clinical Proteomics and Translational Research is mainly focused on the autoimmune neurodegenerative disease multiple sclerosis affecting the central nervous system (CNS) that afflicts young adults leading to paralysis. As the disease doesn’t have any cure, and with an annual economic burden of more than 2 billion dollars, it is important to identify the disease at an early stage and develop new therapies for better disease management. In order to achieve this, we at first developed a density gradient ultracentrifugation methodology to biochemically dissect out myelin and reproduced the in vivo distribution pattern of myelin and myelin-axolemmal proteins in vitro. Combining this technique along with the technique to represent myelin proteins maximally in two-dimensional platform resulted in development of a far-western clinical proteomic technique with which we could identify proteins of clinical significance relating to multiple sclerosis (1,2). The combination led to identification of a variety of molecules of clinical importance which was not identified before and we were able to show the significance of these molecules in the myelin pathology, early diagnosis and therapy towards MS. Most importantly, we demonstrated the role of some of these identified molecules in the pathology of MS, measuring the subclinical activity in MS and its significance in diagnosis of MS (1,3). In addition, we are into development of therapeutically drugs based on structure based drug designing and its validation in animal model of MS, the experimental autoimmune encephalomyelitis along with Dr Gopi Mohan. In an alternate approach towards development of a treatment strategy, we along with Dr Manzoor Koyakutty’s team, have developed a nanoformulation that could reverse the clinical symptoms in animal models of MS. In an extrapolation of the developed technology, we have identified a prognostic marker of clinical significance in central nervous system leukemia of B-acute lymphoblastic leukemia as well. Our lab is also involved in developing biotherapeutics to treat cancers expressing EGFR vIII along with Dr Lakshmi. Thus, overall our aim is to find a strategy to curb autoimmune response in MS and finding alternate prognosis/therapy for different cancers. Dr Menon’s lab has applied for various patents on different findings.

Dr. TESSY XAVIER

Research Associate
Project: CNS leukemia Background: Tessy completed her Masters degree in Biotechnology from Mahatma Gandhi University, Kotayam, India. She Qualified CSIR-JRF and then obtained her doctoral degree in 2015 from the department of Amrita Centre for Nanosciences and Molecular Medicine (ACNSMM), Amrita Viswa Vidyapeetham, India under the supervision of Dr. Krishnakumar Menon. During her PhD she has worked on the identification of novel biomarkers in CNS leukemia. She has also worked as an Assistant research Scientist in the Novartis funded Diabetic Macular Edema project. At present, she is working as a Research Associate in a ICMR funded multi-centre collaborative project in CNS leukemia.

SUNITHA SUBHRAMANIAN

PhD Student
Background: She did her Masters in Bioinformatics from Amrita University, India. Presently working on characterization of oral/subcutaneous efficacy of an inhibitor against a protein which is profoundly found to be over expressed in EAE mice model of multiple sclerosis

SWETHA PALLIKARA

PhD Student
Project: Glial cell factors and their involvement in the pathogenesis of Diabetic Retinopathy Background: She did her Masters in Biotechnology. Before enrolling for PhD, she worked as a Project Fellow in Amrita Centre for Nanosciences and Molecular Medicine. OMPRIYA RATH Junior Research Fellow Background: I have done M.Sc. in neuroscience from Jiwaji University. Subsequently joined as a volunteer in National centre for Autism, NewDelhi. Presently working on Proteomics study upon Epileptic syndrome in Neurochemistry Lab.

MEENAKSHI ANIL

M Tech Molecular Medicine
Background: Meenakshi has completed her Bachelor degree in technology in biotechnology from Sahrdaya College of Engineering and Technology, Thrissur, University of Calicut in 2016. Her Masters Project work was on the influence of multiple sclerosis patient’s cerebrospinal fluid on the proteins involved in inflammation and oxidative stress. She has qualified GATE 2016.

Publications

Publication Type: Journal Article

Year of Publication Title

2019

S. Xavier, Dr. Gopi Mohan C., Shantikumar V Nair, Krishnakumar N. Menon, and Dr. Lakshmi Sumitra, “Generation of Humanized Single-chain Fragment Variable Immunotherapeutic against EGFR Variant III using Baculovirus Expression System and in vitro validation.”, Int J Biol Macromol, vol. 124, pp. 17-24, 2019.[Abstract]


Epidermal growth factor receptor variant III (EGFRvIII) is known to be specifically expressed in cancer cells and associated with tumor virulence. The receptor provides an opportunity for both specifically targeting the tumor cells as well as for potentially controlling and inhibiting tumor progression. In this study, humanized anti-EGFRvIII single-chain fragment variable (hscFv) was expressed in insect cell culture system to accommodate post-translational glycosylations crucial for the fragment stability and efficacy. Target specific binding of the developed fragment to EGFRvIII expressing cell lines and EGFRvIII positive glioblastoma patient samples was evaluated by immunocytochemistry and immunohistochemistry respectively. Downstream intracellular signaling mechanisms related to the action of the developed antibody fragment on growth/metabolism of the cell was evaluated in U87-EGFRvIII human glioblastoma cell lines. It was observed that the hscFv bound specifically to EGFRvIII in mutant expressing cells. Functionally, hscFv was found to confer anti-proliferative properties in EGFRvIII expressing cell lines by downregulating phosphorylation of EGFR/EGFRvIII, Lyn, PI3K and GLUT3 involved in proliferation and metabolism. This study demonstrated the significance of hscFv as a potential immunotherapeutic agent as well as a targeting agent for specific delivery of drugs to EGFRvIII expressing cancer cells.

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2018

S. Xavier, Sundaram, K. R., Biswas, L., Panikar, D., Rajamma, B. M., Nair, S., Krishnakumar N. Menon, and Dr. Lakshmi Sumitra, “Molecular Analysis of Oncogene Expressions in Different Grades of Gliomas”, Journal of Integrated OMICS, vol. 8, pp. 66-73, 2018.[Abstract]


The aggressiveness of brain tumors is attributed to the expression of multiple oncogenes involved in proliferation, metabolism and therapeutic resistance whose potential correlation with tumor progression has not been well-studied. In this study, we aimed to investigate the relationship of oncotargets involved in pathogenesis with respect to glioma grades. Gliomas (n=40) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing for the detection of epidermal growth factor receptor (EGFR) mutants. Expressions levels of EGFRy EGFR variant III (EGFRvIII), Lek/Yes novel tyrosine kinase (Lyn), Spleen tyrosine kinase (SYK), insulin receptor substrate 1 (IRSl)y phosphatidylinositol 3-kinase (PI3K), Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) and glucose transporter 3 (GLUT3) were studied using real-time PCR and compared against glioma grades via statistical methods. Protein expressions were analyzed using immunohistochemistry and western blotting. EGFRvIII was detected in 53% and exon 4 deletion (de4 EGFR) in 20% of gliomas. Importantly, the expressions levels of candidate oncogenes were significantly upregulated (P<0.05) and positively correlated with the glioma grades. Hence, these oncotargets require high surveillance during tumor progression and further investigations on larger patient cohorts can affirm their role as potential markers in the pathology of glioma, thereby aiding in the development of patient-specific multi-targeted therapy. © 2018, Proteomass Scientific Society. All rights reserved.

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2018

S. Kondisetty, Krishnakumar N. Menon, and Pooleri, G. Kumar, “Fibronectin protein expression in renal cell carcinoma in correlation with clinical stage of tumour.”, Biomark Res, vol. 6, p. 23, 2018.[Abstract]


<strong>Background: </strong>Carcinogenesis is a multistep process which involves interplay between the tumour cells and the matrix proteins. This occurs by adherence between the tumour cells and proteins in the extracellular matrix. VHL mutation affects through the hypoxia inducible factor (HIF) and causes changes in various tissue proteins like VEGF, PDGF, TGF, Fibronectin and others. As not much literature is available, we aim to quantify the changes of fibronectin protein in renal cell carcinoma (RCC) tissue.

<strong>Methods: </strong>This Prospective unbalanced case control study was conducted over a period of 18&nbsp;months from April 2016 to September 2017. The patients undergoing nephrectomy for the diagnosis of RCC were included in the study after obtaining written informed consent. Patients were excluded from study, if normal renal tissue could not be identified in the resected kidney and if the artery clamp time to retrieval of tissue was more than 30&nbsp;min. Fibronectin protein is estimated in the tumour tissue by gel electrophoresis and western blotting which is compared with that of normal kidney tissue of the same kidney. Results have been expressed as absolute values with standard deviation and relative expression (RE).

<strong>Results: </strong>Of the 21 patients analysed 15 showed an increase in fibronectin expression in the renal tumour tissue while 6 did not. The mean expression of Fibronectin protein has increased 1.5 times in the tumour tissue when compared with the normal tissue. The increase was 1.54 times in early tumours compared to 1.37 times in advanced tumours of RCC.

<strong>Conclusions: </strong>Fibronectin showed a 1.5 times increase in the tumour compared to normal. This increase is more in Stage 1&amp;2 tumours when compared to the Stage 3&amp;4 tumours.

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2018

S. K. Vettath, Shivashankar, G., Krishnakumar N. Menon, and Dr. Lakshmi Sumitra, “Recombinant Expression of Extracellular Domain of Mutant Epidermal Growth Factor Receptor in Prokaryotic and Baculovirus Expression Systems”, International Journal of Biological Macromolecules, 2018.[Abstract]


Epidermal Growth Factor Receptor variant III (EGFRvIII) is a tumor specific antigen detected in various tumors including gliomas, breast cancer, lung cancer, head and neck squamous cell carcinoma (HNSCC). Screening of EGFRvIII targeting drug molecules can be accelerated by developing drug screening platforms using recombinantly expressed protein. Choice of expression system is one of the major factors deciding the success of recombinant expression of a protein. In our study, we have tried to express and purify the extracellular domain (ECD) of this highly unstable protein using bacterial and baculovirus expression systems to select the expression system suited for our purpose. Even though the protein was successfully expressed in prokaryotic system, purification could be done only under denaturing conditions. But in the baculovirus expression system, the protein was expressed in soluble form and could be purified under native conditions, with single step of purification. Based on our results, we conclude that insect cells are better choice over E. coli cells for expressing EGFRvIII ECD in soluble form. This study provides insights for other researchers involved in expression of similar unstable membrane proteins, on selecting the best expression system and challenges involved.

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2018

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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2016

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.

2016

S. Patil, Tyagi, A., Jose., J., Krishnakumar N. Menon, and Dr. Gopi Mohan C., “Integration of Common Feature Pharmacophore Modeling and in Vitro Study to Identify Potent AChE Inhibitors”, Medicinal Chemistry Research, vol. 25, pp. 2965–2975, 2016.[Abstract]


Alzheimer's disease is a progressive neurodegenerative disorder arising due to genetic and non-genetic causes. One of the major therapies adapted for symptomatic Alzheimer's disease is by targeting acetylcholinesterase enzyme based on the cholinergic hypothesis. Acetylcholinesterase is a substrate-specific enzyme that degrades the neuro-transmitter acetylcholine. An optimum level of acetylcholine should be maintained in the brain for its proper function. In order to identify potent and selective acetylcholinesterase inhibitors we adopted an integrated in silico and bioassay methodologies. In silico approach involves creating chemical features based 3D-pharmacophore models using AChE specific inhibitors. This model was then used for sequential virtual screening from the small molecule databases. Finally, five molecules were selected on the basis of the best docking scores and pharmacokinetics properties. These molecules were subjected to docking analysis with the recently solved crystal structure of human acetylcholinesterase enzyme, in order to reveal its binding mode and interactions at the dual binding sites of the enzyme. The acetylcholinesterase enzyme inhibitory activity of these five lead molecules was further assessed by in-vitro analysis.

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2015

N. Vikkath, Valiyaveedan, S., Nampoothiri, S., Radhakrishnan, N., Pillai, G. S., Nair, V., Pooleri, G. Kumar, Mathew, G., Krishnakumar N. Menon, Ariyannur, P. S., and Pillai, A. B., “Genotype–phenotype analysis of von Hippel–Lindau syndrome in fifteen Indian families”, Familial Cancer, vol. 14, pp. 585–594, 2015.[Abstract]


The general prevalence of the familial multi-organ tumor disorder, von Hippel–Lindau syndrome (VHL), was estimated to be 1 in 25–40,000 in western studies two decades back. Few studies were done in Indian sub-continent, amidst a surge in clinical reports on VHL specific manifestations. The syndrome is correlated with mutations of the gene VHL (located in Chr 3p25.3). We aimed to conduct a prospective case series describing phenotypic and genotypic characteristics in Indian population. The VHL-specific clinical and radiological features were collected from patients and family members. Genotypic changes such as deletion/duplication or point mutation in the VHL locus were identified using sequencing and MLPA. Thirty-one subjects, from fifteen families with diagnosed VHL, were included in the study. Multicystic pancreas was found in 71 {%} (22/31), CNS hemangioblastoma in 68 {%} (21/31), renal cell carcinoma and retinal angiomas in 23 {%} (7/31) each, pheochromocytoma in 9.7 {%} (3/31) of the population and endolymphatic sac tumor in one subject. Four families (9 subjects) had full length deletion of VHL, three families (4 subjects) had a deletion of exon 3, eight families (18 subjects) had different exonic, splice-site and intronic point mutations and one subject had a de novo in-frame indel in exon 1. Multicystic pancreas and CNS hemangioblastomas were the most common manifestations in our population. The phenotypic expression patterns in terms of tumorigenesis, tissue tropism and penetrance in comparison to the genotypic features were found to be different from previous correlative studies.

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2015

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

B. Swarnalatha, Nair, S. L., ,, Paul-Prasanth, B., Krishnakumar N. Menon, Hebbel, R. P., Somani, A., Nair, S. V., Milbauer, L. C., and Jayakumar, R., “Poly (Lactic Acid)-chitosan-Collagen Composite Nanofibers as Substrates for Blood Outgrowth Endothelial Cells”, International Journal of Biological Macromolecules, vol. 58, pp. 220-224, 2013.[Abstract]


In this work, the attachment, viability and functionality of rat Blood Outgrowth Endothelial Cells (rBOEC) and genetically modified rBOEC (rBOEC/eNOS-GFP), which over express endothelial nitric oxide synthase (eNOS), were investigated on Poly(lactic acid) (PLA)-chitosan and PLA-chitosan-collagen nanofibrous scaffolds. Both the cell types displayed good attachment, remained viable and functional on both scaffolds. Moreover, incorporation of collagen in the scaffold helped in sustaining the rBOEC for upto one week, although collagen was not found necessary for rBOEC/eNOS-GFP. We conclude that PLA-chitosan based nanofibrous scaffolds can be a potential candidate for BOEC based wound healing applications. © 2013 Elsevier B.V.

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2013

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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2013

M. P. Narayanan, Dr. Damodaran Vasudevan, and Krishnakumar N. Menon, “Analysis of Gene Mutations among South Indian Patients with Maple Syrup Urine Disease: Identification of Four Novel Mutations”, Indian Journal of Geo-Marine Sciences, vol. 50, pp. 442-446, 2013.[Abstract]


Maple syrup urine disease (MSUD) is predominantly caused by mutations in the BCKDHA, BCKDHB and DBT genes, which encode for the E1α, E1β and E2 subunits of the branched-chain a-keto acid dehydrogenase complex, respectively. Because disease causing mutations play a major role in the development of the disease, prenatal diagnosis at gestational level may have significance in making decisions by parents. Thus, this study was aimed to screen South Indian MSUD patients for mutations and assess the genotype-phenotype correlation. Thirteen patients diagnosed with MSUD by conventional biochemical screening such as urine analysis by DNPH test, thin layer chromatography for amino acids and blood amino acid quantification by HPLC were selected for mutation analysis. The entire coding regions of the BCKDHA, BCKDHB and DBT genes were analyzed for mutations by PCR-based direct DNA sequencing. BCKDHA and BCKDHB mutations were seen in 43% of the total ten patients, while disease-causing DBT gene mutation was observed only in 14%. Three patients displayed no mutations. Novel mutations were c.130C&gt;T in BCKDHA gene, c. 599C&gt;T and c.121_122delAC in BCKDHB gene and c.190G&gt;A in DBT gene. Notably, patients harbouring these mutations were non-responsive to thiamine supplementation and other treatment regimens and might have a worse prognosis as compared to the patients not having such mutations. Thus, identification of these mutations may have a crucial role in the treatment as well as understanding the molecular mechanisms in MSUD. © 2013 National Institute of Science Communication and Information Resources (NISCAIR). All rights reserved.

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2012

A. P. Retnakumari, Hanumanthu, P. L., Malarvizhi, G. L., Prabhu, R., Sidharthan, N., Thampi, M. V., Menon, D., Mony, U., Krishnakumar N. Menon, Keechilat, P., Nair, S., and Dr. Manzoor K., “Rationally Designed Aberrant Kinase-Targeted Endogenous Protein Nanomedicine Against Oncogene Mutated/Amplified Refractory Chronic Myeloid Leukemia”, Molecular Pharmaceutics, vol. 9, pp. 3062-3078, 2012.[Abstract]


Deregulated protein kinases play a very critical role in tumorigenesis, metastasis, and drug resistance of cancer. Although molecularly targeted small molecule kinase inhibitors (SMI) are effective against many types of cancer, point mutations in the kinase domain impart drug resistance, a major challenge in the clinic. A classic example is chronic myeloid leukemia (CML) caused by BCR-ABL fusion protein, wherein a BCR-ABL kinase inhibitor, imatinib (IM), was highly successful in the early chronic phase of the disease, but failed in the advanced stages due to amplification of oncogene or point mutations in the drug-binding site of kinase domain. Here, by identifying critical molecular pathways responsible for the drug-resistance in refractory CML patient samples and a model cell line, we have rationally designed an endogenous protein nanomedicine targeted to both cell surface receptors and aberrantly activated secondary kinase in the oncogenic network. Molecular diagnosis revealed that, in addition to point mutations and amplification of oncogenic BCR-ABL kinase, relapsed/refractory patients exhibited significant activation of STAT5 signaling with correlative overexpression of transferrin receptors (TfR) on the cell membrane. Accordingly, we have developed a human serum albumin (HSA) based nanomedicine, loaded with STAT5 inhibitor (sorafenib), and surface conjugated the same with holo-transferrin (Tf) ligands for TfR specific delivery. This dual-targeted "transferrin conjugated albumin bound sorafenib" nanomedicine (Tf-nAlb-Soraf), prepared using aqueous nanoprecipitation method, displayed uniform spherical morphology with average size of ∼150 nm and drug encapsulation efficiency of ∼74%. TfR specific uptake and enhanced antileukemic activity of the nanomedicine was found maximum in the most drug resistant patient sample having the highest level of STAT5 and TfR expression, thereby confirming the accuracy of our rational design and potential of dual-targeting approach. The nanomedicine induced downregulation of key survival pathways such as pSTAT5 and antiapoptotic protein MCL-1 was demonstrated using immunoblotting. This study reveals that, by implementing molecular diagnosis, personalized nanomedicines can be rationally designed and nanoengineered by imparting therapeutic functionality to endogenous proteins to overcome clinically important challenges like molecular drug resistance. © 2012 American Chemical Society.

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2011

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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2011

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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2010

Krishnakumar N. Menon, Varma, P., Dinesh, K. R., and Raja Biswas, “Role of Probiotic Lactobacillus Plantarum in Inhibiting the Growth and Adhesion of Enteric and Food Borne Pathogens”, J. Food Science, vol. 10, pp. 1750-3841, 2010.

2010

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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Publication Type: Patent

Year of Publication Title

2018

Krishnakumar N. Menon, .Mohan, G., and V., J. Jose, “Composition and Method for Treatment of Diseases Associated with Central Nervous System Inflammation”, U.S. Patent TEMP/E-1/12295/2018-CHE (Patent Filed)2018.

2017

Krishnakumar N. Menon, Kumaran, S., and Kuma, S., “Prognostic Bio-Marker for Multiple Sclerosis and a Subclinical Assessment of Disease Therewith ”, U.S. Patent TEMP/E-1/22385/2017-CHE. (Patent Filed)2017.

2017

, Bernard, C. C. A., Krishnakumar N. Menon, Sadanandan, P., Payne, N. L., and Shantikumar V Nair, “Compositions and Methods for Autoimmune Disease Treatment Inventors”, U.S. Patent Australian Provisional Patent Application no: 2016903294 (Patent Filed)2017.

2014

Krishnakumar N. Menon, Xavier, T., and HARIDASAN, A., “Differential Cerebrospinal Fluid Reactivity to pfdn5-alpha for Detection of B-cell Acute Lymphoblastic Central Nervous System Leukemia”, U.S. Patent PCT/IB2014/058167 Patent Filed2014.

Invited Lectures

  1. “Biochemical dissection of myelin-axolemmal complexes” Biochemistry and Molecular biology symposium, University of Connecticut, USA. 2002.
  2. “Mechanisms in Immune mediated demyelination” Department of Neurology, University of Iowa, Iowa, USA. 2007.
  3. “Biochemical Mechanisms in Demyelination” Department of Biochemistry, Karnataka University, Dharwad, Karnataka, India. 2007.
  4. “"Molecular Mechanisms Involved in the Breakdown of Myelin: Implication for Neurodegenerative Diseases". Raman Imaging Workshop, National Center for Biological Sciences, Bangalore, India. 2007.
  5. "Molecular Mechanisms in Immune-mediated Demyelination". National Brain Research Center, Gurgaon, New Delhi, India. 2007.
  6. “Role of Immune factors in Breakdown of Myelin” Monash Immunology and Stem cell Laboratories, Monash University, Melbourne, Australia. 2008.
  7. “Biochemistry of Myelinolysis” Neural Information Laboratory, National Institute for Physiological Sciences, Okazaki, Japan. 2008.
  8. " Biochemistry behind demyelination and its implications” (2012). Amrita Institute of Medical Sciences, Kochi.
  9. “Identification of novel molecules of clinical significance in neurodegenerative disease using far-western clinical proteomics”. Bioquest International conference (2013). India.
  10. “Far-western clinical proteomics to identify molecules of importance in different autoimmune diseases”. (2014). National Symposium on Trends in Biochemical Sciences. M.S. University of Baroda. India.
  11. “Identification of prognostic marker for central nervous system leukemia and its significance in leukemia pathogenesis” (2015). Next Gen NextGen Genomics, Biology, Bioinformatics and Technology (NGBT) conference, 1-3rd October, HICC, Hyderabad, India
  12. Invited speaker in Amrita samyogam conference on integrative Ayurveda, held at Amrita Institute of Medical Sciences, Kochi. 6-7, August 2017.
  13. Invited speaker in Biological application on Nanoparticles held at IIT Chennai in Dec 4 and 5th 2017. 14. Invited speaker in ICRIMS held at Kochi in July 2018.

Dr. Krishnakumar Menon
Associate Professor, Nanosciences,
Center for Nanosciences, Kochi

krishnakumarmenon@aims.amrita.edu