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.     

Expression of glucocorticoid receptor and glucose transporter-1 during placental development in the diabetic rat

In various tissues, glucocorticoids (GCs) are known to downregulate glucose transport systems; however, their effects on glucose transporters (GLUTs) in the placenta of a diabetic rat are unknown. Glucocorticoid hormone action within the cell is regulated by the glucocorticoid receptor (GR). Thus, this study was designed to investigate the relationship between GR and glucose transporter expression in the placenta of the diabetic rat. Our immunohistochemical results indicated that GR and glucose transporter protein 1 (GLUT 1) are expressed ubiquitously in the trophoblast and endothelial cells of the labyrinthine zone, where maternal fetal transport takes place in the rat placenta. Expression of GR in the junctional zone of the rat placenta was detected in giant cells, and in some spongiotrophoblast cells, but not in the glycogen cells. GLUT 1 was present, especially in glycogen cells during early pregnancy, and in the spongiotrophoblast cells of the junctional zone during late pregnancy. Amounts of GR and GLUT 1 protein were increased towards the end of gestation both in the control and the diabetic placenta. However, at days 17 and 19 of gestation, only the placental GR protein was significantly increased in the streptozotocin-induced diabetic rats compared to control rats. Diabetes led to a significant decrease in placental weight at gestation day 15. In contrast, at gestational days 17 and 21, the weights of the diabetic placenta were significantly increased as compared with the controls. Moreover, diabetes induced fetus intrauterine growth retardation at gestational days 13, 17 and 21. In conclusion, the localization pattern of GR and GLUT 1 proteins in the same cell types led us to believe that there might be a relationship between GR and GLUT 1 expressions at the cellular level. GLUT 1 does not play a pivotal role in diabetic pregnancies. However, placental growth abnormalities during diabetic pregnancy may be related to the amount of GR.     

Evaluation of Copper Bioaccumulation and Translocation in Jatropha curcas Grown in a Contaminated Soil

Contamination of soils with copper (Cu) has become a serious problem in the environment. Phytoremediation is an emerging green technology that uses green plants to remediate heavy metal contaminated areas. This study was conducted to evaluate the potential of Jatropha curcas for remediation of soils contaminated with Cu. Seedlings were planted in soils spiked with Cu in amount of 0, 50, 100, 200, 300, and 400 mg kg^–1 (Cu_0, Cu₅₀,Cu₁₀₀,Cu₂₀₀,Cu_300, and Cu₄₀₀) for a period of five months. The maximum height and number of leaves were recorded in control (Cu₀) whereas the highest basal stem diameter was found in seedlings exposed to Cu₅₀. Copper concentrations among plant parts were in the following trend: roots > stems > leaves. The highest total Cu concentration (665 ± 1 mg kg^?1) and total Cu removal (1.2 ± 0.2%) based on total plant dry biomass were found in Cu₄₀₀ and Cu₅₀ treatments, respectively. J. curcas exhibited high root concentration factor (RCF > 1) and low translocation factor (TF < 1). Although Cu accumulation by the plant didn't reach the criteria of Cu hyperaccumulators, this species showed a potential to be used in phytostabilization of mildly Cu contaminated areas. However, the plant cannot be used for phytoextraction of Cu-contaminated soils.     

Ethanol production from rice hull using Pichia stipitis and optimization of acid pretreatment and detoxification processes

The goal of this study was to produce ethanol from rice hull hydrolysates (RHHs) using Pichia stipitis strains and to optimize dilute acid hydrolysis and detoxification processes by response surface methodology (RSM). The optimized conditions were found as 127.14°C, solid:liquid ratio of 1:10.44 (w/v), acid ratio of 2.52% (w/v), and hydrolysis time of 22.01 min. At these conditions, the fermentable sugar concentration was 21.87 g/L. Additionally, the nondetoxified RHH at optimized conditions contained 865.2 mg/L phenolics, 24.06 g/L fermentable sugar, no hydroxymethylfurfural (HMF), 1.62 g/L acetate, 0.36 g/L lactate, 1.89 g/L glucose, and 13.49 g/L fructose + xylose. Furthermore, RHH was detoxified with various methods and the best procedures were found to be neutralization with CaO or charcoal treatment in terms of the reduction of inhibitory compounds as compared to nondetoxified RHH. After detoxification procedures, the content of hydrolysates consisted of 557.2 and 203.1 mg/L phenolics, 19.7 and 21.60 g/L fermentable sugar, no HMF, 0.98 and 1.39 g/L acetate, 0 and 0.04 g/L lactate, 1.13 and 1.03 g/L glucose, and 8.46 and 12.09 g/L fructose + xylose, respectively. Moreover, the base‐line mediums (control), and nondetoxified and detoxified hydrolysates were used to produce ethanol by using P. stipitis strains. The highest yields except that of base‐line mediums were achieved using neutralization (35.69 and 38.33% by P. stipitis ATCC 58784 and ATCC 58785, respectively) and charcoal (37.55% by P. stipitis ATCC 58785) detoxification methods. Results showed that the rice hull can be utilized as a good feedstock for ethanol production using P. stipitis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:872–882, 2016     

Radionuclides content in grape molasses soil samples from Central Black Sea region of Turkey

The activity concentrations of radionuclides in grape molasses soil samples collected from Zile (Tokat) plain in the Central Black Sea region of Turkey were measured by using gamma spectrometer with a NaI(Tl) detector. Also, the concentrations of ²²²Rn in soil samples and air were estimated essentially taking the activity concentrations of ²²⁶Ra measured in soil samples. Grape molasses soil samples with calcium carbonate content are used for sedimentation for making molasses in this region. The average activity concentrations of ²³²Th, ²²⁶Ra, ⁴⁰K, and ¹³⁷Cs were found as 62 ± 2, 68 ± 3, 479 ± 35, and 8.0 ± 0.3 Bq kg^?1, respectively. The average concentrations of ²²²Rn in soil samples and air were estimated to be 50 kBq m^?3 and 144 Bq m^?3. From the activity concentrations, absorbed gamma dose rate in outdoor air (D), annual effective dose from external exposure (E_E), annual effective dose from inhalation of radon (E_I), and excess lifetime cancer risk (ELCR) were estimated in order to assess radiological risks. The average values of D, E_E, E_I, and ELCR were found to be 90 nGy h^?1, 110 μSv y^?1, 1360 μSv y^?1, and 4 × 10^?4, respectively.     

High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in cells and tissues.

Oligonucleotides that carry a detectable label can be used to probe for mRNA targets in in situ hybridization experiments. Oligonucleotide probes (OPs) have several advantages over cDNA probes and riboprobes. These include the easy synthesis of large quantities of probe, superior penetration of probe into cells and tissues, and the ability to design gene- or allele-specific probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those directly conjugated with horseradish peroxidase (HRP). We used branching phosphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequences to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well both on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also show that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a gene family. This work demonstrates that multihapten-labeled and HRP-conjugated OPs are sensitive and specific and can make superior in situ hybridization probes for both research and diagnostic applications.     

miRNA-375 promotes beta pancreatic differentiation in human induced pluripotent stem (hiPS) cells

Islet transplantation is considered as an ultimate option for the treatment of type I diabetes. Human induced pluripotent stem cells (hiPSCs) have raised the possibility that patient-specific insulin-secreting cells might be derived from somatic cells through cell fate reprogramming. However, current protocols mostly rely on the use of several cytokines and inhibitors for directing differentiation towards pancreatic fate. Given the high manufacturing cost of these recombinant proteins, this approach is prohibitive for clinical applications. Knowing that microRNAs (miRNAs) are key players in various stages of pancreatic development, we present a novel and cost-effective strategy in which over-expression of miR-375 promotes pancreatic differentiation in hiPSCs in the absence of any other stimulator. We used a polycistronic viral vector expressing Sox2, Klf4, c-Myc, and Oct4 to drive hiPSCs from human foreskin fibroblasts. The established hiPSCs are similar to human embryonic stem cells in many aspects including morphology, passaging, surface and pluripotency markers, and gene expression. For differentiation induction, miR-375 was lentivirally overexpressed in these hiPSCs. Morphological assessment, immunocytochemistry, and expression analysis of islet marker genes confirmed that islet like cells were obtained in miR-375 transduced cells compared to controls. Our differentiated clusters secreted insulin in a glucose-dependant manner, showing in vitro functionality. We demonstrated for the first time that miRNAs might be ideal substitutes to induce pancreatic differentiation in hiPSCs. This work provides a new approach to study the role of miRNAs in pancreatic specification and increase the feasibility of using patient-specific iPSCs for beta cell replacement therapy for type I diabetes.