Purification and Characterization of an Active Principle, Lawsone, Responsible for the Plasmid Curing Activity of Plumbago zeylanica Root Extracts

Plasmid curing is the process of obviating the plasmid encoded functions such as antibiotic resistance, virulence, degradation of aromatic compounds, etc. in bacteria. Several plasmid curing agents have been reported in literature, however, no plasmid curing agent can eliminate all plasmids from different hosts. Hence, there is always a need for novel plasmid curing agents that can be effectively used for reversal of plasmid encoded functions such as virulence, antibiotic resistance, etc. In the present study, an active principle responsible for the plasmid curing activity was purified from roots of Plumbago zeylanica by bioassay guided fractionation and identified as 2-hydroxy-1,4-naphthoquinone (lawsone), on the basis of spectral and analytical data such as NMR, GCMS, FTIR. Plasmid curing activity of lawsone was observed against reference as well as wild plasmids (pBR322, pRK2013, R136, pUPI281, and pUPI282) residing in a range of hosts. Curing of plasmid was confirmed by agarose gel electrophoresis. MICs of antibiotics against A. baumannii A24 (pUPI281) and E. coli (pRK2013) decreased significantly in presence of lawsone suggesting synergy between lawsone and antibiotics. Lawsone also inhibited transfer of plasmid pRK2013 to E. coli either by transformation or conjugation. Viability assays (MTT) revealed that lawsone was not toxic to mammalian cells. Thus, the present investigation has revealed lawsone as an effective plasmid curing agent capable of suppressing development and spread of antibiotic resistance. Further, lawsone has important application in basic research to identify phenotypes encoded by the plasmids in plasmid curing experiments. To the best of our knowledge this is the first report of plasmid curing activity of lawsone isolated from roots of P. zeylanica

A Small Molecule Swertisin from Enicostemma littorale

Aim. Stem cell therapy is one of the upcoming therapies for the treatment of diabetes. Discovery of potent differentiating agents is a prerequisite for increasing islet mass. The present study is an attempt to screen the potential of novel small biomolecules for their differentiating property into pancreatic islet cells using NIH3T3, as representative of extra pancreatic stem cells/progenitors. Methods. To identify new agents that stimulate islet differentiation, we screened various compounds isolated from Enicostemma littorale using NIH3T3 cells and morphological changes were observed. Characterization was performed by semiquantitative RT-PCR, Q-PCR, immunocytochemistry, immunoblotting, and insulin secretion assay for functional response in newly generated islet-like cell clusters (ILCC). Reversal of hyperglycemia was monitored after transplanting ILCC in STZ-induced diabetic mice. Results. Among various compounds tested, swertisin, an isolated flavonoid, was the most effective in differentiating NIH3T3 into endocrine cells. Swertisin efficiently changed the morphology of NIH3T3 cells from fibroblastic to round aggregate cell cluster in huge numbers. Dithizone (DTZ) stain primarily confirmed differentiation and gene expression studies signified rapid onset of differentiation signaling cascade in swertisin-induced ILCC. Molecular imaging and immunoblotting further confirmed presence of islet specific proteins. Moreover, glucose induced insulin release (in vitro) and decreased fasting blood glucose (FBG) (in vivo) in transplanted diabetic BALB/c mice depicted functional maturity of ILCC. Insulin and glucagon expression in excised islet grafts illustrated survival and functional integrity. Conclusions. Rapid induction for islet differentiation by swertisin, a novel herbal biomolecule, provides low cost and readily available differentiating agent that can be translated as a therapeutic tool for effective treatment in diabetes

Conditioned Medium Reconditions Hippocampal Neurons against Kainic Acid Induced Excitotoxicity: An In Vitro

Stem cell therapy is gaining attention as a promising treatment option for neurodegenerative diseases. The functional efficacy of grafted cells is a matter of debate and the recent consensus is that the cellular and functional recoveries might be due to “by-stander” effects of grafted cells. In the present study, we investigated the neuroprotective effect of conditioned medium (CM) derived from human embryonic kidney (HEK) cells in a kainic acid (KA) induced hippocampal degeneration model system in in vitro condition. Hippocampal cell line was exposed to KA (200 µM) for 24 hrs (lesion group) whereas, in the treatment group, hippocampal cell line was exposed to KA in combination with HEK-CM (KA + HEK-CM). We observed that KA exposure to cells resulted in significant neuronal loss. Interestingly, HEK-CM cotreatment completely attenuated the excitotoxic effects of KA. In HEK-CM cotreatment group, the cell viability was ~85–95% as opposed to 47% in KA alone group. Further investigation demonstrated that treatment with HEK-CM stimulated the endogenous cell survival factors like brain derived neurotrophic factors (BDNF) and antiapoptotic factor Bcl-2, revealing the possible mechanism of neuroprotection. Our results suggest that HEK-CM protects hippocampal neurons against excitotoxicity by stimulating the host’s endogenous cell survival mechanisms

Islet-Like Cell Aggregates Generated from Human Adipose Tissue Derived Stem Cells Ameliorate Experimental Diabetes in Mice

BACKGROUND: Type 1 Diabetes Mellitus is caused by auto immune destruction of insulin producing beta cells in the pancreas. Currently available treatments include transplantation of isolated islets from donor pancreas to the patient. However, this method is limited by inadequate means of immuno-suppression to prevent islet rejection and importantly, limited supply of islets for transplantation. Autologous adult stem cells are now considered for cell replacement therapy in diabetes as it has the potential to generate neo-islets which are genetically part of the treated individual. Adopting methods of islet encapsulation in immuno-isolatory devices would eliminate the need for immuno-suppressants. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we explore the potential of human adipose tissue derived adult stem cells (h-ASCs) to differentiate into functional islet like cell aggregates (ICAs). Our stage specific differentiation protocol permit the conversion of mesodermic h-ASCs to definitive endoderm (Hnf3β, TCF2 and Sox17) and to PDX1, Ngn3, NeuroD, Pax4 positive pancreatic endoderm which further matures in vitro to secrete insulin. These ICAs are shown to produce human C-peptide in a glucose dependent manner exhibiting in-vitro functionality. Transplantation of mature ICAs, packed in immuno-isolatory biocompatible capsules to STZ induced diabetic mice restored near normoglycemia within 3-4 weeks. The detection of human C-peptide, 1155±165 pM in blood serum of experimental mice demonstrate the efficacy of our differentiation approach. CONCLUSIONS: h-ASC is an ideal population of personal stem cells for cell replacement therapy, given that they are abundant, easily available and autologous in origin. Our findings present evidence that h-ASCs could be induced to differentiate into physiologically competent functional islet like cell aggregates, which may provide as a source of alternative islets for cell replacement therapy in type 1 diabetes

Can yoga therapy stimulate stem cell trafficking from bone marrow?

It has been established that mesenchymal stromal cells (MSCs) from bone marrow enter the peripheral circulation intermittently for possible tissue regeneration, repair and to take care of daily wear and tear. This is evident from the detection of MSCs from peripheral blood. The factors governing this migration remain elusive. These MSCs carry out the work of policing and are supposed to repair the injured tissues. Thus, these cells help in maintaining the tissue and organ homeostasis. Yoga and pranayama originated in India and is now being practiced all over the world for positive health. So far, the chemical stimulation of bone marrow has been widely used employing injection of colony stimulating factor. However, the role of physical factors such as mechanical stimulation and stretching has not been substantiated. It is claimed that practicing yoga delays senescence, improves the physiological functions of heart and lung and yoga postures make the body elastic. It remains to be seen whether the yoga therapy promotes trafficking of the stem cells from bone marrow for possible repair and regeneration of worn out and degenerating tissues. We cover in this short review, mainly the role of physical factors especially the yoga therapy on stem cells trafficking from bone marrow

Shell-Less Chick Embryo Culture as an Alternative in vitro Model to Investigate Glucose-Induced Malformations in Mammalian Embryos

We have developed a simple shell-less chick embryo culture system to study glucose-induced malformations. This system involves the culturing of chick embryos from the second day to the fifth day of incubation, with associated yolk and thick and thin albumen outside the egg shell. The system allows the observation of embryonic development of chicks in a glass bowl. Developing embryos at 24 h, 48 h and 72 h incubation, corresponding to the Hamberger Hamilton (HH) stages from 7 to 21, were treated with two concentrations of glucose (50 mM and 100 mM) for 24 h. Glucose treatment resulted in a mortality rate of over 70% in younger embryos. Furthermore, a variety of malformations such as retarded growth, abnormal heart development, macrosomia, exencephaly, etc. were observed in older embryos, which were similar to those reported in mammalian embryos as a consequence of diabetic pregnancy. The glucose-induced malformations were found to be concentration- and stage-dependent, thus emphasizing the roles of the degree of hyperglycemia and the stage of embryonic development in diabetic growth anomalies. Here we demonstrate for the first time that the present system can be used (i) for experiments at early stages of chick embryo development and (ii) for assessing the effects of acute glucose toxicity similar to those reported for mammalian embryos in a hyperglycemic environment

Approaches Towards Endogenous Pancreatic Regeneration

The phenomenon of pancreatic regeneration in mammals has been well documented. It has been shown that pancreatic tissue is able to regenerate in several species of mammal after surgical insult. This tissue is also known to have the potential to maintain or increase its β-cell mass in response to metabolic demands during pregnancy and obesity. Since deficiency in β-cell mass is the hallmark of most forms of diabetes, it is worthwhile understanding pancreatic regeneration in the context of this disease. With this view in mind, this article aims to discuss the potential use in clinical strategies of knowledge that we obtained from studies carried out in animal models of diabetes. Approaches to achieve this goal involve the use of biomolecules, adult stem cells and gene therapy. Various molecules, such as glucagon-like peptide-1, β-cellulin, nicotinamide, gastrin, epidermal growth factor-1 and thyroid hormone, play major roles in the initiation of endogenous islet regeneration in diabetes. The most accepted hypothesis is that these molecules stimulate islet precursor cells to undergo neogenesis or to induce replication of existing β-cells, emphasizing the importance of pancreas-resident stem/progenitor cells in islet regeneration. Moreover, the potential of adult stem cell population from bone marrow, umbilical cord blood, liver, spleen, or amniotic membrane, is also discussed with regard to their potential to induce pancreatic regeneration