Contributions

 

Human Haematopoietic stem cell Laboratory

 In vitro differentiations of cultured Human Haematopoietic stem cells into different cell lineages

 

 

 Stem cell therapy offers a great potential in the treatment of several diseases and it becomes an attractive basis for cell-based regenerative therapies. Haematopoietic stem cells (HSCs) are characterized by the presence of CD34+ and CD 133+ cell surface proteins; they inhabit in bone marrow. In our department human CD34+ cells were isolated from peripheral blood of a donor was mobilized with G-CSF (5µg/kg/day) using AsTech cell separator fitted with Rvy kit. Thus isolated CD34+ cells through Apheresis technique were evaluated by Immuno histochemical, immunoblot and FACS techniques. These pure cells were cultured in DMEM containing 10% FBS, 5% CO₂ at 37^°C and with 95% humidity. With the help of growth factors and other lineage specific factors we were able to differentiate these human HSCs into cardiomyocytes, astrocytes, oligodendrocytes, type II pneumocytes, osteocytes, podocytes, Beta cells of islets of langerhans, hepatocytes, erythrocytes, megakaryocytes leading to functional platelets, indicating that human haematopoietic stem cells have the ability to form any type cells of human body. In this process developing different lineages from HSCs novel methodologies for differentiation were developed in the department and of them two methodologies have been submitted for obtaining patents (1. Indian patent filed: TR-5 CBR No. 13582/E-2/1374/2019-CHE. Application number: 201941016975. 2. Indian patent filed: TR-5 CBR No. 13583/E-2/1375/2019-CHE. Application number: 201941016976). Autologous transplantation of these HSCs could be the best option to regenerate the damaged tissues and organs. Standardizing such lineages differentiated from HSCs became very useful in understanding in the pathophysiology of diseases involving these differentiated cells. For example, endongeneous erythroid colony formation assay (CFU-E assay) is used to understand the formation of erythrocytes from HSCs. This has become very important parameter in the diagnosis of erythrocytosis.

 Medical genetics Laboratory

                             

DNA and RNA isolations, PCR, qPCR and mutational analysis of various diseases

 

Medical genetics is any application of genetic principles to medical practice. This includes studies of inheritance, mapping disease genes, diagnosis and treatment, and genetic counselling. We are actively involved in the genetic analysis of various genes involved or implicated in different diseases and assisting our faculty of various departments in getting information in this regard.  In this process we have identified several novel alleles in several genes which are implicated in the progression of the disease or in the diagnosis of the disease. Some of the important genes in which novel mutations were identified are:

Polycystic kidney disease 1 (PKD1), Angiotensin I converting enzyme (ACE), Von Hippel- Lindau tumor suppressor  (VHL), Wilm’s tumor 1 (WT1), Complement Factor H (CFH), Proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1/CD2BP1), ATPase V-type subunit B1 (ATP6V1B1), Glucokinase (GCK), BRCA1, BRCA2, MGMT, NF-2, AKT, NPHS1,  BCR-abl ,  mbCOMT,  EPO-R, EPASI, EGLN1 and VHL genes

Recently, we have identified novel alleles in the kinase domain of BCR-abl gene which was contributing in the imatinib resistance in CML patients. This study was undertaken in collaboration with Department of Haematology. Further, causative reason for erythrocytosis (both Polycythemia vera and congenital erythrocytosis) have been answered through genetic analysis of JAK2 (exons 12 and 14), EPASI, EPO-R and VHL genes wherein we have identified reported and novel alleles respectively, this study is also carried with active cooperation of Department of Haematology. 

 

 

                                                 

                     BCR-ABL  novel alleles                                           mbCOMT gene novel alleles

Microbial Genetics Laboratory

                     

Development of novel drugs against Staphylococcus aureus

                                          

Staphylococcus aureus is a common inhabitant of human nasopharynx. It is also a cause of life-threatening illness, producing a potent array of virulence factors that enable survival in normally sterile sites. The transformation of S. aureus from commensal to pathogen is poorly understood. Up-regulation of virulence factors aids in mature biofilm formation which is metabolically regulated the expressions of IDH, LDH, PK, NADK, IMPDH, SDH and STPK regulates the redox status in the pathogen paving way for the expression of virulence factors and mature biofilm formation which are the key pathogenic factors of S. aureus. In house enzyme assays and biofilm tests were developed to understand the involvement of metabolism in the expression of virulence factors and biofilm formation. In this process spectrophotometric analysis of STPK and BYK were developed and this unique process has been patented (Indian Patent filed: TR-5. C.B.R. NO: 6535).

Occurrence of multidrug resistance strains of S. aureus has complicated the treatment and management of S. aureus infections and new drug targets which are pathogen specific is the need of the hour. In this perspective we have developed MMOXC (4-methoxy-1-methyl-2-oxopyridine-3-carbamide) a derivative of ricinine MMOXC and a novel Dibenzyl (benzo[d] thiazol-2-yl(hydroxy)methyl) phosphonate (3b) derived from α-Hydroxyphosphonate  which exhibited prominent anti-S. aureus and anti-biofilm properties by blocking cell wall formation, RNA biosynthesis and protein maturation.