Transplantation of stromal-derived factor 1α and basic fibroblast growth factor primed insulin-producing cells reverses hyperglycaemia in diabetic rats.

The defective insulin production is associated with severely reduced islet cell mass leading to diabetes. Growth factors preconditioned stem cells have arisen as an effective therapy to treat many diseases including diabetes. The current study was designed to assess the effect of pretreatment of ASCs derived IPCs with combination of stromal cell derived factor 1 alpha (SDF1α) and basic fibroblast growth factor (bFGF) in improving glucose tolerance in streptozotocin induced diabetic rats. The results showed maximally significant reduction in hyperglycaemia and fibrosis, while up-regulation of survival and pancreas-specific genes, insulin levels and homing of transplanted cells in SDF-1α + bFGF IPCs transplanted rats as compared with other groups. Moreover, increased expression of insulin, glucagon and Glut-2 in pancreas of the SDF-1α + bFGF IPCs transplanted group indicated more regeneration of pancreas. Hence, the use of IPCs preconditioned with SDF-1α + bFGF would be more effective for treating diabetes.

Bioactivity studies of Huh-7 cells derived human epidermal growth factor expressed in Pichia pastoris.

Previously, we have reported cloning of human epidermal growth factor gene from Huh-7 cells and its extracellular expression in Pichia pastoris. The presented work is a detailed report regarding molecular characterization of Huh-7 cells-derived hEGF expressed in Pichia pastoris with special reference to its glycosylation profiling and bioactivity studies. Densitometric scanning of SDS-PAGE separated extracellular proteins from hEGF recombinant Pichia pastoris strain indicated that about 84% of the extracellular proteins were glycosylated. Size exclusion chromatography using Superdex 75 prep grade column was successfully utilized to separate fractions containing glycosylated and non-glycosylated extracellular proteins. In dot blot assay, hEGF was detected in both glycosylated and non-glycosylated fractions. Bioactivity assays revealed that both glycosylated and non-glycosylated fractions were bioactive as determined by cell viability assay. It was also observed that hEGF present in non-glycosylated fraction was relatively more bioactive than hEGF present in glycosylated fraction.

Diazoxide preconditioning of endothelial progenitor cells from streptozotocin-induced type 1 diabetic rats improves their ability to repair diabetic cardiomyopathy.

Type 1 diabetes mellitus (DM) is a strong risk factor for the development of diabetic cardiomyopathy (DCM) which is the leading cause of morbidity and mortality in the type 1 diabetic patients. Stem cells may act as a therapeutic agent for the repair of DCM. However, deteriorated functional abilities and survival of stem cells derived from type 1 diabetic subjects need to be overcome for obtaining potential outcome of the stem cell therapy. Diazoxide (DZ) a highly selective mitochondrial ATP-sensitive K(+) channel opener has been previously shown to improve the ability of mesenchymal stem cells for the repair of heart failure. In the present study, we evaluated the effects of DZ preconditioning in improving the ability of streptozotocin-induced type 1 diabetes affected bone marrow-derived endothelial progenitor cells (DM-EPCs) for the repair of DCM in the type 1 diabetic rats. DM-EPCs were characterized by immunocytochemistry, flow cytometry, and reverse transcriptase PCR for endothelial cell-specific markers like vWF, VE cadherin, VEGFR2, PECAM, CD34, and eNOS. In vitro studies included preconditioning of DM-EPCs with 200 µM DZ for 30 min followed by exposure to either 200 µM H2O2 for 2 h (for oxidative stress induction) or 30 mM glucose media (for induction of hyperglycemic stress) for 48 h. Non-preconditioned EPCs with and without exposure to H2O2 and 30 mM high glucose served as controls. These cells were then evaluated for survival (by MTT and XTT cell viability assays), senescence, paracrine potential (by ELISA for VEGF), and alteration in gene expression [VEGF, stromal derived factor-1α (SDF-1α), HGF, bFGF, Bcl2, and Caspase-3]. DZ preconditioned DM-EPCs demonstrated significantly increased survival and VEGF release while reduced cell injury and senescence. Furthermore, DZ preconditioned DM-EPCs exhibited up-regulated expression of prosurvival genes (VEGF, SDF-1α, HGF, bFGF, and Bcl2) on exposure to H2O2, and VEGF and Bcl2 on exposure to hyperglycemia while down regulation of Caspase-3 gene. Eight weeks after type 1 diabetes induction, DZ preconditioned, and non-preconditioned DM-EPCs were transplanted into left ventricle of diabetic rats (at a dose of 2 × 10(6) DM-EPCs/70 µl serum free medium). After 4 weeks, DZ preconditioned DM-EPCs transplantation improved cardiac function as assessed by Millar's apparatus. There was decrease in collagen content estimated by Masson's trichrome and sirius red staining. Furthermore, reduced cell injury was observed as evidenced by decreased expression of Caspase-3 and increased expression of prosurvival genes Bcl2, VEGF, and bFGF by semi-quantitative real-time PCR. In conclusion, the present study demonstrated that DZ preconditioning enhanced EPCs survival under oxidative and hyperglycemic stress and their ability to treat DCM.

Multiple metal tolerance and biosorption of cadmium by Candida tropicalis isolated from industrial effluents: glutathione as detoxifying agent.

The ability of cadmium uptake by metal-resistant yeast, Candida tropicalis, from the liquid medium and wastewater was evaluated. The minimum inhibitory concentration of Cd(2+) against C. tropicalis was 2,500 mg L(-1). The yeast also showed tolerance toward Zn(2+) (1,400 mg L(-1)), Ni(2+) (1,000 mg L(-1)), Hg(2+) (1,400 mg L(-1)), Cu(2+) (1,000 mg L(-1)), Cr(6+) (1,200 mg L(-1)), and Pb(2+) (1,000 mg L(-1)). The yeast isolate showed typical growth curves, but lag and log phases extended in the presence of cadmium. The yeast isolate showed optimum growth at 30°C and pH 8. The metal processing ability of the isolate was determined in a medium containing 100 mg L(-1) of Cd(2+). C. tropicalis could decline Cd(2+) 70%, 85%, and 92% from the medium after 48, 96, and 144 h, respectively. C. tropicalis was also able to remove Cd(2+) 40% and 78% from the wastewater after 6 and 12 days, respectively. Cd produced an increase in glutathione (GSH) and nonprotein thiol levels by 135% and 134% at 100-mg L(-1) concentration, respectively. An increase in the synthesis of GSH is involved in metal tolerance, and the presence of increasing GSH concentrations may be a marker for high metal stress in C. tropicalis. C. tropicalis, which is resistant to heavy metal ions and is adaptable to the local environmental conditions, may be employed for metal detoxification operations.

PFSA DNA database: A tool to hunt the serial offenders.

A forensic DNA database comprises of thousands of DNA profiles generated from suspects, convicts or even from common people from society. It is used for the cross-matching of DNA profiles obtained from evidence items collected from a crime scene. These databases are playing a core role in clearing the innocent and solving the dead-end unresolved crimes ultimately leading to crime reduction. In March, 2017, a nine years old minor girl was raped in district Khushab (Province Punjab). The medico-legal examiner indicated brutal sexual violence on the victim. Police apprehended a suspect who was excluded as the source of foreign male DNA from tested evidence items. Thus the case put up a question mark on the capabilities and efficiency of the police. An unknown male DNA profile obtained from evidence items was uploaded to PFSA DNA Database to maintain record. Later on, a suspect was arrested by Karachi (Province Sindh) police in another rape case, DNA profile of suspect was searched in the PFSA DNA Database. This generated DNA profile matched with the foreign DNA profile obtained from evidence items of minor victim, hence this atrocious crime was resolved. PFSA DNA Database provides support to criminal prosecution and also leads to identify potential suspects. It took years of effort to develop the rich PFSA DNA Database which subsequently proved to be fruitful in the exoneration of innocents and conviction of offenders in criminal cases.

Assessment of metals induced histopathological and gene expression changes in different organs of non-diabetic and diabetic rats.

Diabetes is a complex metabolic disorder and different environmental toxicants including heavy metals have been involved in diabetes induction. Therefore, assessment of the environmental risk factors and heavy metals induced toxicity have become critical for reducing the consequences of metals pollutants. Previously, we reported heavy metals induced nephrotoxicity in non-diabetic and diabetic rats. Here, we extended our analysis by examining the heavy metals induced organs (heart, kidney, liver, pancreas, and spleen) damage in diabetic and non-diabetic Wistar rats using histopathology and quantitative real-time PCR (qRT-PCR). Following the generation of the diabetic rat model, the animals were exposed to heavy metals including lead (Pb), arsenic (As), manganese (Mn) and cadmium (Cd). Both non-diabetic and diabetic rats were exposed to heavy metals for 30 days and subsequently, the heart, kidney, liver, pancreas and spleen tissues were examined. Heavy metal treatment resulted in irregularly arranged myofibrils and vacuolization in the heart tissue of metal treated groups as evident from hematoxylin and eosin (H & E) staining. The kidney tissue of rats treated with heavy metals showed tubular degeneration, fibrosis, hemorrhage, and vacuolation. The liver of the heavy metals treated rats exhibited cellular degeneration and necrosis. The pancreatic tissue of streptozotocin injected untreated and metal treated rats revealed severe degeneration, necrosis, degranulation, shrinkage, and depression in the islets of Langerhans. Increased red pulp area and congestion were observed in the spleen of the metal mixture treated non-diabetic and diabetic rats. In line with the histological data, the qRT-PCR analysis showed downregulated expression of Bcl2 and upregulation of Caspase-3 in non-diabetic and diabetic metal treated rats as compared to the non-diabetic untreated rats. In conclusion, the present study revealed, diabetic rats are more prone to metal alone as well as metal mixture induced organ damage as compared to non-diabetic rats.

Metal-induced nephrotoxicity to diabetic and non-diabetic Wistar rats.

The present study was conducted to examine the nephrotoxic effects of heavy metals including lead (Pb), manganese (Mn), arsenic (As), and cadmium (Cd) in diabetic and non-diabetic Wistar rats. Animals were exposed to heavy metals for 30 days, Pb was injected as lead acetate (C4H6O4Pb), Mn was injected as manganese chloride (MnCl2), Cd was injected as cadmium chloride (CdCl2), and As was administered orally to rats in the form of sodium arsenite (AsO2Na). Results showed that metal deposition trends in tissues were Pb > As > Cd > Mn and the urinary metal levels were Pb > Cd > As > Mn. Diabetic metal alone, as well as metal mixture-treated groups, showed decreased urinary metal levels as compared with non-diabetic metal alone and metal mixture-treated groups. Both diabetic- and non-diabetic metal mixture-treated groups revealed an increasing trend of blood urea nitrogen (BUN) and serum creatinine. In addition, heavy metal treatments resulted in elevated malondialdehyde (MDA) levels in the kidney tissue while decreased levels of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione (GHS) were observed in the kidney tissue in comparison with the control group. The histological analysis of the kidney tissues showed tubular degeneration, fibrosis, and vacuolation as a result of heavy metal exposure. The present study revealed that co-exposure of heavy metals (Pb, Cd, Mn, As) induced more nephrotoxicity as compared with the metal alone treatment. Moreover, diabetic Wistar rats are more prone to kidney damage as a result of heavy metal exposure.

In vitro preconditioning of insulin-producing cells with growth factors improves their survival and ability to release insulin.

Glucose-induced oxidative stress in the diabetic pancreas directly affects viability and the consequent therapeutic outcome of transplanted stem cells. Pretreatment of stem cells with growth factors induces tolerance in them against various stresses (hypoxia, thermal or hyperglycaemic). This study investigated the effect of pretreatment on insulin-producing cells (IPCs) differentiated from adipose-derived mesenchymal stem cells (ADMSCs), with a combination of stromal cell-derived factor 1 alpha (SDF1 α) and basic fibroblast growth factor (bFGF) against hyperglycaemic stress (17 or 33 mM glucose). The results showed that IPCs pretreated with a combination of SDF1α and bFGF exhibited maximally alleviated apoptosis, senescence and cell damage with a concomitantly increased release of insulin, enhanced cell proliferation and greater upregulation of Insulin 1, Insulin 2, Ngn3, Pdx1 and Nkx6.2 when stressed with 33 mM glucose. These findings may offer an improved therapeutic outcome for the treatment of diabetes.

Protective role of vitamin E preconditioning of human dermal fibroblasts against thermal stress in vitro.

AIMS: Oxidative microenvironment of burnt skin restricts the outcome of cell based therapies of thermal skin injuries. The aim of this study was to precondition human dermal fibroblasts with an antioxidant such as vitamin E to improve their survival and therapeutic abilities in heat induced oxidative in vitro environment. MAIN METHODS: Fibroblasts were treated with 100µM vitamin E for 24h at 37°C followed by heat shock for 10min at 51°C in fresh serum free medium. KEY FINDINGS: Preconditioning with vitamin E reduced cell injury as demonstrated by decreased expression of annexin-V, cytochrome p450 (CYP450) mediated oxidative reactions, senescence and release of lactate dehydrogenase (LDH) accomplished by down-regulated expression of pro-apoptotic BAX gene. Vitamin E preconditioned cells exhibited remarkable improvement in cell viability, release of paracrine factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), stromal derived factor-1alpha (SDF-1α) and also showed significantly up-regulated levels of PCNA, VEGF, BCL-XL, FGF7, FGF23, FLNß and Col7α genes presumably through activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. SIGNIFICANCE: The results suggest that pretreatment of fibroblasts with vitamin E prior to transplantation in burnt skin speeds up the wound healing process by improving the antioxidant scavenging responses in oxidative environment of transplanted burn wounds.