Kalamegam Gauthaman

Derivation and differentiation of bone marrow mesenchymal stem cells from osteoarthritis patients

Osteoarthritis (OA) of the knee is a degenerative joint disease caused by the progressive reduction of the articular cartilage surface that leads to reduced joint function. Cartilage degeneration occurs through gradual loss in extracellular matrix components including type II collagen and proteoglycan. Due to limited inherent self repair capacity of the cartilage, the use of cell-based therapies for articular cartilage regeneration is considered promising. Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent cells and are highly capable of multilineage differentiation which render them valuable for regenerative medicine. In this study, BM-MSCs were isolated from OA patients and were characterized for MSC specific CD surface marker antigens using flowcytometry and their differentiation potential into adipocytes, osteocytes and chondrocytes were evaluated using histological and gene expression studies. BM-MSCs isolated from OA patients showed short spindle shaped morphology in culture and expressed positive MSC related CD markers. They also demonstrated positive staining with oil red O, alizarin red and alcian blue following differentiation into adipocytes, osteocytes and chondrocytes, respectively. In addition, chodrogenic related genes such as collagen type II alpha1, cartilage oligomeric matrix protein, fibromodulin, and SOX9 as well as osteocytic related genes such as alkaline phosphatase, core-binding factor alpha 1, osteopontin and RUNX2 runt-related transcription factor 2 were upregulated following chondrogenic and osteogenic differentiation respectively. We have successfully isolated and characterized BM-MSCs from OA patients. Although BM-MSCs has been widely studied and their potential in regenerative medicine is reported, the present study is the first report in our series of experiments on the BMSCs isolated from OA patients at King Abdulaziz University Hospital, Jeddah, Saudi Arabia.

Identification of Unique miRNA Biomarkers in Colorectal Adenoma and Carcinoma Using Microarray: Evaluation of Their Putative Role in Disease Progression

MicroRNAs (miRNAs) are known to be dysregulated and play a key role in cancer progression. The present study aims to identify the miRNAs associated with colorectal adenoma and carcinoma to evaluate their role in tumor progression and metastasis using microarray. In silico analysis of miRNAs was performed on five different microarray data sets that represented the genes and miRNAs expressed in colorectal adenoma and carcinoma. We identified 10 different miRNAs that were common to both colorectal adenoma and carcinoma, namely, miR9, miR96, miR135b, miR137, miR147, miR182, miR183, miR196b, miR224, and miR503. Of these, miR135b and miR147 were significantly downregulated in colorectal adenoma but upregulated in carcinoma. In addition, we studied the gene expression profile associated with colorectal adenocarcinoma and identified three genes, namely, ZBED3, SLC10A3, and FOXQ1, that were significantly downregulated in colorectal adenoma compared to carcinoma. Interestingly, of all the miRNAs and genes associated with colorectal adenocarcinoma, the myoglobin (MB) gene was identified to be under the direct influence of miR135b, showing an inverse relationship between them in adenoma and carcinoma. Most of the identified miRNAs and associated genes are involved in signaling pathways of cell proliferation, angiogenesis, and metastasis. The present study has identified putative miRNA targets and their associated gene networks which could be used as potential biomarkers of colon adenocarcinoma. Moreover, the association of miR135b and MB gene is very unique and can be considered as a lead candidate for novel cancer therapeutics.

Identification of novel genetic variations affecting osteoarthritis patients

BackgroundOsteoarthritis (OA) is a progressive joint disease characterized by gradual degradation of extracellular matrix (ECM) components in the cartilage and bone. The ECM of cartilage is a highly specified structure that is mainly composed of type II collagen and provides tensile strength to the tissue via aggrecan and proteoglycans. However, changes in the ECM composition and structure can lead to loss of collagen type II and network integrity. Several risk factors have been correlated with OA including age, genetic predisposition, hereditary factors, obesity, mechanical injuries, and joint trauma. Certain genetic association studies have identified several genes associated with OA using genome-wide association studies (GWASs).ResultsWe identified several novel genetic variants affecting genes that function in several candidate causative pathways including immune responses, inflammatory and cartilage degradation such as SELP, SPN, and COL6A6.ConclusionsThe approach of whole-exome sequencing can be a promising method to identify genetic mutations that can influence the OA disease.

Biology of human primitive erythroblasts for application in noninvasive prenatal diagnosis

Human primitive erythroblasts produced during early embryogenesis have been found in maternal circulation at early gestation and are considered good target cells for noninvasive prenatal diagnosis. We aimed to gain a better understanding of the biology of primitive erythroblasts and maximize their potential utility for noninvasive prenatal diagnosis.

Encapsulation of 5-Flurouracil into PLGA Nanofibers and Enhanced Anticancer Effect in Combination with Ajwa-Dates-Extract (Phoenix dactylifera L.)

Side effects connected with chemotherapeutic agents used in cancer treatment has led to alternative modalities of combinatorial therapies in an attempt to reduce the drug dosage and associated risks. In the current study we evaluated the potential use of Ajwa Dates Extract (ADE), reported to have anti-cancer effects, as an adjuvant therapy in combination with 5-flurouracil (5FU) against the human-breast-adenocarcinoma cell line (MFC-7) in vitro. The effects of ADE alone and in combination with 5-FU were evaluated in terms of cell viability and cytotoxicity. For drug delivery purpose, we successfully encapsulated 5FU in both presence and absence of ADE through electrospinning together with poly lactic-co-glycolic acid (PLGA) in different combinations. Physicochemical properties of 5FU and ADE incorporated into PLGA nanofibers remained unaltered as confirmed by Fourier-Transform-Infrared (FTIR), Raman-spectroscopies and X-ray Diffraction (XRD) techniques. The morphological characterization of nanofibers was done using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The surface roughness of PLGA and PLGA + ADE nanofibers increased by incorporation of 5FU. PLGA + ADE nanofibers were in hydrophilic range (<90°) while nanofibers prepared from both PLGA + 5FU and PLGA + 5FU + ADE combinations were in hydrophobic range (∼112°). The percentage inhibition of MCF-7 proliferation at 72 hrs showed an enhanced combinatorial anti-cancer effect of 5FU and ADE on the cells seeded on PLGA + 5FU + ADE mat (47% decrease) while PLGA + 5FU and PLGA + ADE demonstrated only 23% and 16% decrease respectively as compared to controls. The hydrophobicity induced by 5FU can further be investigated to get improved cellular adherence and efficient controlled-drug-release.

In-Silico Characterization and in-Vivo Validation of Albiziasaponin-A, Iso-Orientin, and Salvadorin Using a Rat Model of Alzheimer's Disease

Alzheimers disease (AD) is a neurodegenerative disorder characterized by dementia, excessive acetylcholinesterase (AChE) activity, formation of neurotoxic amyloid plaque, and tau protein aggregation. Based on literature survey, we have shortlisted three important target proteins (AChE, COX2, and MMP8) implicated in the pathogenesis of AD and 20 different phytocompounds for molecular docking experiments with these three target proteins. The 3D-structures of AChE, COX2, and MMP8 were predicted by homology modeling by MODELLER and the threading approach by using ITASSER. Structure evaluations were performed using ERRAT, Verify3D, and Rampage softwares. The results based on molecular docking studies confirmed that there were strong interactions of these phytocompounds with AChE, COX2, and MMP8. The top three compounds namely Albiziasaponin-A, Iso-Orientin, and Salvadorin showed least binding energy and highest binding affinity among all the scrutinized compounds. Post-docking analyses showed the following free energy change for Albiziasaponin-A, Salvadorin, and Iso-Orientin (−9.8 to −15.0 kcal/mol) as compared to FDA approved drugs (donepezil, galantamine, and rivastigmine) for AD (−6.6 to −8.2 Kcal/mol) and interact with similar amino acid residues (Pro-266, Asp-344, Trp-563, Pro-568, Tyr-103, Tyr-155, Trp-317, and Tyr-372) with the target proteins. Furthermore, we have investigated the antioxidant and anticholinesterase activity of these top three phytochemicals namely, Albiziasaponin-A, Iso-Orientin, and Salvadorin in colchicine induced rat model of AD. Sprague Dawley (SD) rat model of AD were developed using bilateral intracerebroventricular (ICV) injection of colchicine (15 μg/rat). After the induction of AD, the rats were subjected to treatment with phytochemicals individually or in combination for 3 weeks. The serum samples were further analyzed for biomarkers such as 8-hydroxydeoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE), tumor necrosis factor-alpha (TNF-α), cyclooxygenase-2 (COX-2), matrix metalloproteinase-8 (MMP-8), isoprostanes-2 alpha (isoP-2α), and acetylcholine esterase (AChE) using conventional Enzyme Linked Immunosorbent Assay (ELISA) method. Additionally, the status of lipid peroxidation was estimated calorimetrically by measuring thiobarbituric acid reactive substances (TBARS). Here, we observed a statistically significant reduction (P < 0.05) in the oxidative stress and inflammatory markers in the treatment groups receiving mono and combinational therapies using Albiziasaponin-A, Iso-Orientin, and Salvadorin as compared to colchicine alone group. Besides, the ADMET profiles of these phytocompounds were very promising and, hence, these potential neuroprotective agents may further be taken for preclinical studies either as mono or combinational therapy for AD.

Stem Cells in Bone and Articular Cartilage Tissue Regeneration

Multiple factors including trauma, infection, ageing, obesity and tumours result in bone and cartilage defects. The regeneration and functional restoration of bone and cartilage remains a significant clinical challenge. ‘Autologous grafts’ continue to remain the ‘gold standard’ in both bone and cartilage regeneration but stem cell-based therapies offer great promise in both these areas. Despite the plethora of stem cells that exist within the human body, the challenge remains in identifying the most beneficial cell type, assessing their availability, expansion under cGMP culture conditions, differentiation potential and functional restoration capacity. Embryonic stem cells; mesenchymal stem cells from the bone marrow, synovial fluid, adipose tissue and umbilical cord; and primary articular chondrocytes are some of the candidate cell types that are extensively studied in the context of bone (and cartilage) regeneration. The limited regeneration potential of cartilage adds further complexity to cartilage tissue engineering compared to the bone. However, major bone reconstruction as in the case of large bone defects due to tumour resection, fractures, and skeletal deformities is equally challenging. Incorporation of novel biomaterials, understanding the optimal cell-scaffold interactions, the addition of growth factors and provision of molecular cues are all essential in achieving effective tissue regeneration. Intensive effects in tissue regeneration can actually predispose to tissue hypertrophy, which also limits functional capacity. The current state of-the-art in both bone and cartilage regeneration is reviewed in this chapter, which highlights the importance of combined approaches involving stem/progenitor cells, biomolecules and/or biomaterials for therapies as well as rehabilitation and improvement in quality of life.

Disease associated cellular machinery in anaphylaxis - And the de novo paradigm shift

Anaphylaxis is a sudden immune reaction against an allergen that can potentially lead to Anaphylactic Shock (AS). This immune reaction is characterized by an increase in Immunoglobulin-E (IgE) type of antibodies that bind with FcεRI receptors on mast cells to release inflammatory mediators. Various intracellular signaling molecules downstream of IgE/ FcεRI axis play a potential role in cytokine, chemokine and eicosanoid secretion as well as degranulation of immune cells causing vasodilation, vascular permeability, and reduction of intravascular volume leading to cardiovascular collapse. Here, we discuss the cellular machinery of anaphylaxis and the de novo paradigm shift in the cellular aspects of AS.

Molecular docking of resveratrol with known protein structures as well as high-throughput meta-data analyses uncork the “French Paradox” and its potential “Druggable” targets in humans

Background Resveratrol (RSV) is a polyphenolic phytoalexin (3, 5, 4’trihydroxystilbene) produced by plants as a protective defense mechanism against pathogens or environmental stress [1]. RSV is found in various plant species such as peanuts, mulberries, Japanese knotweed and skin of grapes. As such RSV is a component of red wine. Interestingly, the prevalence of coronary heart disease (CHD) is comparatively reduced in people from Southern France despite high dietary intake of saturated fats [1-3]. In order to uncork this “French-Paradox”, as well as to identify other potential health benefits of RSV in relation to cancer, infection and inflammation, we studied molecular docking of RSV with known protein structures implicated in health and disease and further dissected the potential molecular networks regulated by RSV.