Dental stem cell banking: Techniques and protocols

Dental tissue-derived stem cells (DSCs) provide an easy, accessible, relatively noninvasive promising source of adult stem cells (ASCs), which brought encouraging prospective for their clinical applications. DSCs provide a perfect opportunity to apply for a patient's own ASC, which poses a low risk of immune rejection. However, problems associated with the long-term culture of stem cells, including loss of proliferation and differentiation capacities, senescence, genetic instability, and the possibility of microbial contamination, make cell banking necessary. With the rapid development of advanced cryopreservation technology, various international DSC banks have been established for both research and clinical applications around the world. However, few studies have been published that provide step-by-step guidance on DSCs isolation and banking methods. The purpose of this review is to present protocols and technical details for all steps of cryopreserved DSCs, from donor selection, isolation, cryopreservation, to characterization and quality control. Here, the emphasis is on presenting practical principles in accordance with the available valid guidelines.     

Efficient electrocatalysis of L-cysteine oxidation at carbon ionic liquid electrode

The electrochemistry of L-cysteine (CySH) in neutral aqueous media was investigated using carbon ionic liquid electrode (CILE). Comparative experiments were carried out using glassy carbon electrodes. At CILE, highly reproducible and well-defined cyclic voltammograms were obtained for l-cysteine with a peak potential of 0.49V vs Ag/AgCl, showing that CILE manifests a good electrocatalytic activity toward oxidation of l-cysteine. A linear dynamic range of 2-210microM with an experimental detection limit of 2microM was obtained. The method was successfully applied to the determination of l-cysteine in a sample of soya milk. Cysteine oxidation at CILE does not result in deactivation of the electrode surface. Mechanistic studies showed that, at CILE, the overall CySH oxidation is controlled by the oxidation of the CyS(-) electroactive species.     

Successive subculturing alters spore-bound Pr1 activity,germination and virulence of the entomopathogenic fungus,Beauveria bassiana

One of the hurdles in the development of entomopathogenic fungi such as Beauveria bassiana is loss of virulence when successively maintained in vitro. This may result in products of inferior quality in mass production programs. Also, there are many contradicting data and unclear points in this case. Three isolates of B. bassiana were subcultured successively 15 times. Spore-bound Pr1 activity, germination rate, and virulence of conidia against mealworm (Tenebrio molitor L.) larvae were studied. Results showed that isolates normally retained their virulence during 10 subculturings. However, they clearly offered decreased virulence (elevated LT₅₀ values and lower percent mortality). The activity of Pr1 bound to conidia declined as subculturing continued; the lowest spore-bound activity and germination potential of conidia was recorded for the 15th subculture. Virulence data were in agreement with Pr1 activity and germination rate as there was a positive correlation between germination rate and spore-bound Pr1 activity with fungal virulence. This explains that at least a part of attenuation in fungal virulence can be explored in enzymatic activity, especially in the important cuticle-degrading protease, Pr1.     

Electrodeposition of gold-platinum alloy nanoparticles on ionic liquid-chitosan composite film and its application in fabricating an amperometric cholesterol biosensor

An electrodeposition method was applied to form gold-platinum (AuPt) alloy nanoparticles on the glassy carbon electrode (GCE) modified with a mixture of an ionic liquid (IL) and chitosan (Ch) (AuPt-Ch-IL/GCE). AuPt nanoparticles were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and electrochemical methods. AuPt-Ch-IL/GCE electrocatalyzed the reduction of H(2)O(2) and thus was suitable for the preparation of biosensors. Cholesterol oxidase (ChOx) was then, immobilized on the surface of the electrode by cross-linking ChOx and chitosan through addition of glutaraldehyde (ChOx/AuPt-Ch-IL/GCE). The fabricated biosensor exhibited two wide linear ranges of responses to cholesterol in the concentration ranges of 0.05-6.2 mM and 6.2-11.2 mM. The sensitivity of the biosensor was 90.7 μA mM(-1) cm(-2) and the limit of detection was 10 μM of cholesterol. The response time was less than 7 s. The Michaelis-Menten constant (K(m)) was found as 0.24 mM. The effect of the addition of 1 mM ascorbic acid and glucose was tested on the amperometric response of 0.5 mM cholesterol and no change in response current of cholesterol was observed.     

Rare BRCA1 haplotypes including 3′UTR SNPs associated with breast cancer risk

Genetic markers identifying women at an increased risk of developing breast cancer exist, yet the majority of inherited risk remains elusive. While numerous BRCA1 coding sequence mutations are associated with breast cancer risk, BRCA1 mutations account for less then 5% of breast cancer risk. Since 3′ untranslated region (3′UTR) polymorphisms disrupting microRNA (miRNA) binding can be functional and can act as genetic markers of cancer risk, we tested the hypothesis that such polymorphisms in the 3′UTR of BRCA1 and haplotypes containing these functional polymorphisms may be associated with breast cancer risk. We sequenced the BRCA1 3′UTR from breast cancer patients to identify miRNA disrupting polymorphisms. We further evaluated haplotypes of this region including the identified 3′UTR variants in a large population of controls and breast cancer patients (n = 221) with known breast cancer subtypes and ethnicities. We identified three 3′UTR variants in BRCA1 that are polymorphic in breast cancer populations, and haplotype analysis including these variants revealed that breast cancer patients harbor five rare haplotypes not generally found among controls (9.50% for breast cancer chromosomes, 0.11% for control chromosomes, p = 0.0001). Three of these rare haplotypes contain the rs8176318 BRCA1 3′UTR functional variant. These haplotypes are not biomarkers for BRCA1 coding mutations, as they are found rarely in BRCA1 mutant breast cancer patients (1/129 patients = 0.78%). These rare BRCA1 haplotypes and 3′UTR SNPs may represent new genetic markers of breast cancer risk.Key words: BRCA1, haplotype, microRNA, SNP, 3′UTR, breast cancer, triple negative breast cancer     

Human Dietary Exposure to Fumonisin B1 from Iranian Maize Harvested During 1998–2000

Fumonisin B₁ (FB₁) is the most abundant of the fumonisin mycotoxins, mainly produced in maize by F. verticillioides and F. proliferatum. A previous study on the FB1 contamination of maize harvested in Mazandaran and Isfahan Provinces of Iran in 1998 and 1999 demonstrated contamination in both provinces. This present study was undertaken to further investigate the variation in levels of contamination and to estimate possible levels of human exposure to fumonisins in Iran. The mean level of FB₁ in 49 visually healthy maize samples collected from Mazandaran Province during 2000 was 6.14 mg/kg, which is higher than that found during 1998 and 1999 (2.27 and 3.18 mg/kg, respectively). Although these levels are higher than the Iranian legislative limits for fumonisins in maize intended for humans, the relatively low estimated consumption of maize in Iran (3.3 g/person/day) implies that average exposures (0.011 and 0.215 μg/kg body weight/day in Isfahan and Mazandaran, respectively) are within the provisional maximum tolerable daily intake of 2 μg/kg body weight/day set by the Joint FAO/WHO Expert Committee on Food Additives. Nevertheless, certain sections of the population who may consume higher amounts of maize or who may replace all or some of their consumption of other cereals with maize, could well exceed this limit.     

Simultaneous determination of dopamine, ascorbic acid, and uric acid using carbon ionic liquid electrode

A recently constructed carbon composite electrode using room temperature ionic liquid as pasting binder was employed as a novel electrode for sensitive, simultaneous determination of dopamine (DA), ascorbic acid (AA), and uric acid (UA). The apparent reversibility and kinetics of the electrochemical reaction for DA, AA, and UA found were improved significantly compared to those obtained using a conventional carbon paste electrode. The results show that carbon ionic liquid electrode (CILE) reduces the overpotential of DA, AA, and UA oxidation, without showing any fouling effect due to the deposition of their oxidized products. In the case of DA, the oxidation and reduction peak potentials appear at 210 and 135mV (vs Ag/AgCl, KCl, 3.0M), respectively, and the CILE shows a significantly better reversibility for dopamine. The oxidation peak due to the oxidation of AA occurs at about 60mV. For UA, a sharp oxidation peak at 340mV and a small reduction peak at 250mV are obtained at CILE. Differential pulse voltammetry was used for the simultaneous determination of ternary mixtures of DA, AA, and UA. Relative standard deviation for DA, AA, and UA determinations were less than 3.0% and DA, AA, and UA can be determined in the ranges of 2.0x10(-6)-1.5x10(-3), 5.0x10(-5)-7.4x10(-3), and 2.0x10(-6)-2.2x10(-4)M, respectively. The method was applied to the determination of DA, AA, and UA in human blood serum and urine samples.     

Fabrication of a glucose sensor based on a novel nanocomposite electrode

The direct electrocatalytic oxidation of glucose in alkaline medium at nanoscale nickel hydroxide modified carbon ionic liquid electrode (CILE) has been investigated. Enzyme free electro-oxidation of glucose have greatly been enhanced at nanoscale Ni(OH)(2) as a result of electrocatalytic effect of Ni(+2)/Ni(+3) redox couple. The sensitivity to glucose was evaluated as 202 microA mM(-1)cm(-2). From 50 microM to 23 mM of glucose can be selectively measured using platelet-like Ni(OH)(2) nanoscale modified CILE with a detection limit of 6 microM (S/N=3). The nanoscale nickel hydroxide modified electrode is relatively insensitive to electroactive interfering species such as ascorbic acid (AA), and uric acid (UA) which are commonly found in blood samples. Long-term stability, high sensitivity and selectivity as well as good reproducibility and high resistivity towards electrode fouling resulted in an ideal inexpensive amperometric glucose biosensor applicable for complex matrices.     

Effects of type of binder and conducting phase on the performance of solid‐state electrochemiluminescence composites

The electrochemiluminescence (ECL) of tris(2,2‐bipyridyl)ruthenium [Ru(bpy)₃]²⁺ has received much attention. By immobilizing [Ru(bpy)₃]²⁺ on an electrode surface, solid‐state ECL has several advantages over solution‐phase ECL, such as reduced amounts of costly reagent and a simplified experimental design. Herein, different types of solid‐state ECL sensors were fabricated and the performances of paraffin oil and two ionic liquids (ILs) as the binders were compared for the construction of solid‐state ECL. Scanning electron microscopy (SEM), CCD camera, UV–vis, fluorescence spectroscopy, electrochemistry and ECL were applied to characterize and evaluate the performance of the solid‐state composites. According to the obtained results, Ru–graphite/IL octyl pyridinium hexaflurophosphate (OPPF₆) was introduced as a new solid‐state ECL with excellent properties such as simple preparation, low background current, fast electron‐transfer rate and good reproducibility and stability. Moreover, for a study of the effect of carbon structure on the performance of the electrode, graphite was replaced by multi‐walled carbon nanotubes (MWCNTs) and Ru–MWCNT/OPPF₆ was constructed and its efficiency was compared with Ru–graphite/OPPF₆ composite electrode. Copyright © 2013 John Wiley & Sons, Ltd.