I. Stem Cells and Tissue Engineering

 

 

 In vitro differentiation of cultured human CD34+ cells into different cell types

 

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 are characterized by the presence of CD34+ and CD 133+ cell surface proteins; they inhabit in bone marrow and other tissues. These cells in bone marrow are involved in the formation of blood and cells of immune system. Recently, their ability to differentiate into other multiple tissue lineages has encouraged using them in the treatment of various debilitating diseases. 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 and immunoblot techniques using anti CD34 monoclonal antibody (QBEND 10 clone). These cells were cultured in DMEM containing 10% FBS, 5% CO₂ at 37^°C and with 95% humidity. With the help of growth factors Like EGF, FGF and initiating factors at various concentrations we have achieved the development of astrocytes, cardiomyocytes and pneumocytes cell types which are of ectoderm, mesoderm, and endoderm origin. These astrocytes, cardiomyocytes, pneumocytes and osteocytes shows the specific markers GFAP, GLUL, AQP4, S100b, GATA4, MLC2V, SP-B, SP-C, RUNX-2, OSTERIX, SPARK and SCLEROSTIN expressions were confirmed through RT-PCR. Autologous transplantation of these CD34 cells at sites of injuries has shown significant improvement in regaining the functioning of tissues and organs. However, transplantation of such differentiated cells may hasten the outcome and could be the futuristic approach in curing the diseases.

II. Microbial Genetics

 

 

Cloning, expression and characterization of various genes of Staphylococcus aureus involved in Pathogenesis

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 regulated by expressions of IDH, LDH, PK, NADK, IMPDH, SDH and STPK.  The results obtained clearly indicate that expression of these enzymes is essential for the upregulation of virulence factors and mature biofilm formation which are the key pathogenic factors of S. aureus.  Occurrence of multidrug resistance strains of S. aureus has complicated the treatment and management of S .aureus infections. In this perspective Peptidyl deformylase (def), Penta peptide ligase (murF), and Urdine monophosphosphate kinase (UMPK) appears to be novel drug targets which are involved in protein synthesis, peptidoglycan, cell wall and RNA biosynthesis respectively all these features are the important factors in the formation mature biofilms. Therefore we propose inhibitors of these enzymes would be appropriate and effective antimicrobial agents against S. aureus infections.

III. Medical genetics

 

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

Characterized genes

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