A voltage-sensitive dye-based assay for the identification of differentiated neurons derived from embryonic neural stem cell cultures

Background

Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca²⁺-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate.

Methodology/Principal Findings

In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters.

Conclusions/Significance

Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture.     

Netrin-1 and Semaphorin 3A Predict the Development of Acute Kidney Injury in Liver Transplant Patients

Acute kidney injury (AKI) is a serious complication after liver transplantation. Currently there are no validated biomarkers available for early diagnosis of AKI. The current study was carried out to determine the usefulness of the recently identified biomarkers netrin-1 and semaphorin 3A in predicting AKI in liver transplant patients. A total of 63 patients’ samples were collected and analyzed. AKI was detected at 48 hours after liver transplantation using serum creatinine as a marker. In contrast, urine netrin-1 (897.8±112.4 pg/mg creatinine), semaphorin 3A (847.9±93.3 pg/mg creatinine) and NGAL (2172.2±378.1 ng/mg creatinine) levels were increased significantly and peaked at 2 hours after liver transplantation but were no longer significantly elevated at 6 hours after transplantation. The predictive power of netrin-1, as demonstrated by the area under the receiver-operating characteristic curve for diagnosis of AKI at 2, 6, and 24 hours after liver transplantation was 0.66, 0.57 and 0.59, respectively. The area under the curve for diagnosis of AKI was 0.63 and 0.65 for semaphorin 3A and NGAL at 2 hr respectively. Combined analysis of two or more biomarkers for simultaneous occurrence in urine did not improve the AUC for the prediction of AKI whereas the AUC was improved significantly (0.732) only when at least 1 of the 3 biomarkers in urine was positive for predicting AKI. Adjusting for BMI, all three biomarkers at 2 hours remained independent predictors of AKI with an odds ratio of 1.003 (95% confidence interval: 1.000 to 1.006; P = 0.0364). These studies demonstrate that semaphorin 3A and netrin-1 can be useful early diagnostic biomarkers of AKI after liver transplantation.     

The very many faces of presenilins and the γ-secretase complex

Presenilin is a central, catalytic component of the γ-secretase complex which conducts intramembrane cleavage of various protein substrates. Although identified and mainly studied through its role in the development of amyloid plaques in Alzheimer disease, γ-secretase has many other important functions. The complex seems to be evolutionary conserved throughout the Metazoa, but recent findings in plants and Dictyostelium discoideum as well as in archeons suggest that its evolution and functions might be much more diversified than previously expected. In this review, a selective survey of the multitude of functions of presenilins and the γ-secretase complex is presented. Following a brief overview of γ-secretase structure, assembly and maturation, three functional aspects are analyzed: (1) the role of γ-secretase in autophagy and phagocytosis; (2) involvement of the complex in signaling related to endocytosis; and (3) control of calcium fluxes by presenilins.     

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.     

Polymorphism of VDR gene--the most effective molecular marker of osteoporotic bone fractures risk within postmenopausal women from Wielkopolska region of Poland

The major public health problem which will arise is a frequency of osteoporosis. The first manifestations of this disease are often bone fractures. Identification and evaluation of individual bone fracture risk will be the most effective way of solving the problem. Genetic determination of osteoporosis is unquestionable. The aim of this study is to detect which variants of genotypes lead to illness. We investigated 187 patients with osteoporosis (161 women, 26 men) and 19 healthy subjects. Polymorphisms of the following genes were investigated: OPG, VDR, ESR1, TGFB1 COL1A1, and BMP2. The statistically significant relationship between BMD value and T allele of Taq I VDR gene were found. Genotypes: aa, bb, TT of VDR gene occur more frequently in polish osteoporotic population in Wielkopolska region within patients with higher risk of bone fractures.     

Next-generation sequencing of representational difference analysis products for identification of genes involved in diosgenin biosynthesis in fenugreek (Trigonella foenum-graecum)

Planta - Representational difference analysis of cDNA was performed and differential products were sequenced and annotated. Candidate genes involved in biosynthesis of diosgenin in fenugreek were...     

Kanamycin induces free radicals formation in melanocytes: An important factor for aminoglycosides ototoxicity

Ototoxicity is well-documented but not fully understood undesirable side effect of aminoglycoside antibiotic, kanamycin. Kanamycin is capable of binding to melanin biopolymers—natural pigments of the skin, hair, and eyes. Melanin-producing cells, melanocytes, are also present in the inner ear and are known to be necessary for normal hearing. It was considered that melanin content in the inner ear may influence aminoglycoside-induced ototoxic effect. The impact of kanamycin on melanocytes homeostasis may thus play role in the antibiotic-induced ototoxic effect. Previously, we demonstrated that kanamycin disturbs homeostasis in light-pigmented melanocytes. To investigate if/how melanization contributes to this phenomenon, the study using in vitro model of dark-pigmented melanocytes is required. Spectrophotometric measurements and electron paramagnetic resonance (EPR) spectroscopy analysis were performed. Kanamycin induced a concentration-dependent loss in HEMn-DP melanocytes viability. The value of IC ₅₀ was estimated to be 5.0 mM. Modulation of the activity of analyzed antioxidant enzymes and increased production of free radicals as well as the decrease of the melanin content were observed. Our results confirmed that kanamycin generates oxidative stress in melanocytes. The increased level of free radicals caused by kanamycin may be responsible for the imbalance of antioxidant defense and the reduction of melanin content in melanocytes. The role of melanin in the mechanism of kanamycin-induced hearing impairment was discussed and the obtained results were compared with the previously demonstrated data concerning light-pigmented melanocytes.     

Impact of Mushrooms’ Vegetative Places and Morphological Parts of a Fruiting Body on the Fatty Acids Profile of Wild Leccinum aurantiacum and Leccinum versipelle

The aim of this study was to indicate potential differences in composition of fatty acids between two mushroom species as well as to examine the impact of mushrooms’ vegetative places and morphological parts of a fruiting body on the fatty acids profile. The research material consisted of 72 samples of wild Leccinum aurantiacum and Leccinum versipelle in the form of caps and stipes, collected from three selected regions of Poland. Determination of the examined compounds was performed by gas chromatography (FID). Linoleic (C18 : 2), oleic (C18 : 1) and palmitic (C16:0) acids were the predominant compounds in all samples under study. The profile of fatty acids in Leccinum aurantiacum and Leccinum versipelle was varied depending on mushroom species, a region and morphological parts of a fruiting body. The high content of polyunsaturated fatty acids in polish L. aurantiacum and L. versipelle provides that the mushroom may be recommended in different types of diets.     

Protein phosphorylation associated with stimulation of rabbit gastric glands

Changes in protein phosphorylation associated with stimulation of acid secretion were investigated using isolated rabbit gastric glands labeled with 32P. The glands were stimulated by 100 microM histamine plus either 10 microM forskolin or 50 microM isobutylmethylxanthine, homogenized, and fractionated into a series of pellets: 40 X g, 5 min; 4000 X g, 10 min; 14,500 X g, 10 min; 48,200 X g, 90 min (microsomes); and supernatant. Stimulation induced a redistribution of H+/K+-transporting ATPase among the membrane fractions, i.e., a reduction in activity of the microsomal fraction, and a compensatory increase in the 4000 X g fraction. Further subfractionation of the 4000 X g pellet by Ficoll density gradient produced an 18% Ficoll layer, greatly enriched in the H+/K+-ATPase, and which is thought to be rich in apical membranes of parietal cells. SDS-polyacrylamide gel electrophoresis showed that the amount of 94 kDa peptide (the molecular size of the H+/K+-ATPase) was increased in the 18% Ficoll layer and decreased in the microsomal fraction by stimulation. Analysis of autoradiograms of the gels revealed that apparent changes in level of phosphorylation occurred in the 120, 94 and 80 kDa regions of the 18% Ficoll layer, and in the 94 kDa region of the microsomal fraction. The phosphorylation changes in the 94 kDa region may not reflect changes in specific activity of a single peptide but may be due to the heterogeneity of proteins in this region, which was demonstrated by selective heat treatment of the samples as well as two-dimensional electrophoresis. Phosphorylation of 120 kDa protein in the 18% Ficoll layer was clearly increased by stimulation, and this appeared to be associated with protein distribution changes as well as phosphorylation. The 80 kDa protein in the 18% Ficoll layer showed marked increased phosphorylation by stimulation, with little change in protein distribution. This 80 kDa protein was focused on two-dimensional gels as several sequential spots, with the most radioactive peptide focused toward the acidic side; thus, we propose isomeric forms of an 80 kDa protein with sequential phosphorylation sites. The phosphorylation changes observed in this study are considered to be important to the process of gastric acid secretion because they occurred in the putative apical membrane fractions in which biochemical and functional changes with stimulation have been demonstrated.