Induced in vitro differentiation of neural-like cells from human exfoliated deciduous teeth-derived stem cells

Stem cells from human exfoliated deciduous teeth (SHED) are highly proliferative, clonogenic and multipotent stem cells with a neural crest cell origin. Additionally, they can be collected with minimal invasiveness in comparison with other sources of mesenchymal stem cells (MSCs). Therefore, SHED could be a desirable option for potential therapeutic applications. In this study, SHEDs were established from enzyme-disaggregated deciduous dental pulp obtained from 6 to 9 year-old children. The cells had typical fibroblastoid morphology and expressed antigens characteristic of MSCs, STRO1, CD146, CD45, CD90, CD106 and CD166, but not the hematopoietic and endothelial markers, CD34 and CD31, as assessed by FACS analysis. Differentiation assessment revealed a strong osteogenic and adipogenic potential of SHEDs. In order to further evaluate the in vitro differentiation potential of SHED into neural cells, a simple short time growth factor-mediated induction was used. Immunofluorescence staining and flow cytometric analysis revealed that SHED rapidly expressed nestin and b-III tubulin, and later expressed intermediate neural markers. In addition, the intensity and percentages of nestin and b-III tubulin and mature neural markers (PSA-NCAM, NeuN, Tau, TH, or GFAP) increased significantly following treatment. Moreover, RT-PCR and Western blot analyses showed that the neural markers were strongly up-regulated after induction. In conclusion, these results provide evidence that SHED can differentiate into neural cells by the expression of a comprehensive set of genes and proteins that define neural-like cells in vitro. SHED cells might be considered as new candidates for the autologous transplantation of a wide variety of neurological diseases and neurotraumatic injuries.     

Differentiation of human embryonic stem cells to hepatocyte-like cells on a new developed xeno-free extracellular matrix

Human embryonic stem cells (hESCs) provide a new source for hepatocyte production in translational medicine and cell replacement therapy. The reported hESC-derived hepatocyte-like cells (HLCs) were commonly generated on Matrigel, a mouse cell line-derived extracellular matrix (ECM). Here, we performed the hepatic lineage differentiation of hESCs following a stepwise application of growth factors on a newly developed serum- and xeno-free, simple and cost-benefit ECM, designated “RoGel,” which generated from a modified conditioned medium of human fibroblasts. In comparison with Matrigel, the differentiated HLCs on both ECMs expressed similar levels of hepatocyte-specific genes, secreted α-fetoprotein, and metabolized ammonia, showed glycogen storage activity as well as low-density lipoprotein and indocyanine green uptake. The transplantation of hESC–HLCs into the carbon tetrachloride-injured liver demonstrated incorporation of the cells into the host mouse liver and the expression of albumin. The results suggest that the xeno-free and cost-benefit matrix may be applicable in bioartificial livers and also may facilitating a clinical application of human pluripotent stem cell-derived hepatocytes in the future.     

A reliable and efficient protocol for inducing genetically transformed roots in medicinal plant Nepeta pogonosperma

Nepeta pogonosperma is an important medicinal plant with anti-inflammatory effects. An efficient and reliable transformation system for this plant was developed through optimization of several factors which affected the rate of Agrobacterium rhizogenes mediated transformation. Five bacterial strains, A4, ATCC15834, LBA9402, MSU440 and A13, two explant types, leaves and stems, and several co-cultivation media were examined. The maximum rate of hairy root induction was obtained from stem explants using MSU440 and ATCC15834 bacterial strains. A drastic increase in the frequency of transformation (91 %) was observed when MS medium lacking NH₄NO₃, KH₂PO₄, KNO₃ and CaCl₂. Hairy root lines were confirmed by polymerase chain reaction (PCR) using primers of the rolB gene. According to Southern blot analysis, one T-DNA copy was inserted into each of the hairy root lines. In the present study, transgenic hairy roots have been obtained trough genetic transformation by A. rhizogenes harbouring two plasmids, the Ri plasmid and pBI121 binary vector harbouring gus reporter gene. Expression of the gus gene in transgenic hairy root was confirmed by histochemical GUS assay.     

High Amylose Resistant Starch Diet Ameliorates Oxidative Stress,Inflammation, and Progression of Chronic Kidney Disease

Inflammation is a major mediator of CKD progression and is partly driven by altered gut microbiome and intestinal barrier disruption, events which are caused by: urea influx in the intestine resulting in dominance of urease-possessing bacteria; disruption of epithelial barrier by urea-derived ammonia leading to endotoxemia and bacterial translocation; and restriction of potassium-rich fruits and vegetables which are common sources of fermentable fiber. Restriction of these foods leads to depletion of bacteria that convert indigestible carbohydrates to short chain fatty acids which are important nutrients for colonocytes and regulatory T lymphocytes. We hypothesized that a high resistant starch diet attenuates CKD progression. Male Sprague Dawley rats were fed a chow containing 0.7% adenine for 2 weeks to induce CKD. Rats were then fed diets supplemented with amylopectin (low-fiber control) or high fermentable fiber (amylose maize resistant starch, HAM-RS2) for 3 weeks. CKD rats consuming low fiber diet exhibited reduced creatinine clearance, interstitial fibrosis, inflammation, tubular damage, activation of NF_kB, upregulation of pro-inflammatory, pro-oxidant, and pro-fibrotic molecules; impaired Nrf2 activity, down-regulation of antioxidant enzymes, and disruption of colonic epithelial tight junction. The high resistant starch diet significantly attenuated these abnormalities. Thus high resistant starch diet retards CKD progression and attenuates oxidative stress and inflammation in rats. Future studies are needed to explore the impact of HAM-RS2 in CKD patients.     

Comparison of the Role of D1- and D2-Like Receptors in the CA1 Region of the Hippocampus in the Reinstatement Induced by a Subthreshold Dose of Morphine and Forced Swim Stress in Extinguished Morphine-CPP in Rats

Reward-seeking and relapse to drug use are two characteristics of addiction and reports have indicated the role of hippocampal structures in reward learning. To find the best ways of treatment, the understanding of the neurobiological mechanisms of reward and its involved factors is a must. For this reason, in the present study, we aimed to investigate the role of D1- and D2-like dopamine receptors and compared their activities in the CA1 region, focusing on the reinstatement induced by forced swim stress (FSS) or the combination of FSS and a subthreshold dose of morphine in extinguished morphine-CPP in rats. The rats were bilaterally implanted by two separate cannulas into the CA1 region. The animals received different doses of SCH23390 or sulpiride (0.5, 2, and 4 µg/0.5 µl vehicle/side) into the CA1 region on the reinstatement day and were tested for FSS-induced reinstatement or the combination of FSS and a subthreshold dose of morphine in separate groups. Our findings indicated that the D1- and D2-like receptor antagonists attenuated the reinstatement induced by the combination of FSS and the subthreshold dose of morphine. The behavioral results were more prominent in the groups of animals that received SCH23390 as compared to sulpiride. The data may suggest a role for the dopamine receptors in the CA1 region in relapse to drugs of abuse, which may be induced by exposure to a stressor.     

Molecular Epidemiology of P. vivax in Iran: High Diversity and Complex Sub-Structure Using Neutral Markers,but No Evidence of Y976F Mutation at pvmdr1

BackgroundMalaria remains endemic at low levels in the south-eastern provinces of Iran bordering Afghanistan and Pakistan, with the majority of cases attributable to P. vivax. The national guidelines recommend chloroquine (CQ) as blood-stage treatment for uncomplicated P. vivax, but the large influx of imported cases enhances the risk of introducing CQ resistance (CQR).ConclusionsThe contrasting low versus high diversity in the two sub-populations (K1 and K2) infers that a combination of local transmission and cross-border malaria from higher transmission regions shape the genetic make-up of the P. vivax population in south-eastern Iran. There was no molecular evidence of CQR amongst the local or imported cases, but ongoing clinical surveillance is warranted.     

ADP-ribosylation is involved in the integration of foreign DNA into the mammalian cell genome.

The most commonly used DNA transfection method, which employs the calcium phosphate co-precipitation of the donor DNA, involves several discrete steps (1,2). These include the uptake of the donor DNA by the recipient cells, the transport of the DNA to the nucleus, transient expression prior to integration into the host cell genome, concatenation and integration of the transfected DNA into the host cell genome and finally the stable expression of the integrated genes (2,3). Both the concatenation and the integration of the donor DNA into the host genome involve the formation and ligation of DNA strand-breaks. In the present study we demonstrate that the nuclear enzyme, adenosine diphosphoribosyl transferase (ADPRT, E.C. 2.4.2.30), which is dependent on the presence of DNA strand breaks for its activity (4,5) and necessary for the efficient ligation of DNA strand-breaks in eukaryotic cells (4,6), is required for the integration of donor DNA into the host genome. However, ADPRT activity does not influence the uptake of DNA into the cell, its episomal maintenance or replication, nor its expression either before or after integration into the host genome. These observations strongly suggest the involvement of ADPRT activity in eukaryotic DNA recombination events.