Neural differentiation of human umbilical cord matrix-derived mesenchymal cells under special culture conditions

Many neural disorders are characterized by the loss of one or several types of neural cells. Human umbilical cord-derived mesenchymal cells (hUCMs) are capable of differentiating into neuron, astroglia-like and oligodendrocyte cell types. However, a reliable means of inducing the selective differentiation of hUCMs into neural cells in vitro has not yet been established. For induction of neural differentiation, hUCMs were seeded onto sterile glass slides and six various cocktails using a base medium (DMEM/LG) supplemented with 10 % FBS, retinoic acid (RA), dimethyl sulfoxide (DMSO), epidermal growth factor (EGF) and fibroblast growth factor (FGF) were used to compare their effect on neuronal, astrocyte and oligodandrocyte differentiation. The hUCMs were positive for mesenchymal markers, while they were negative for hematopoietic markers. Differentiation to adipogenic and osteogenic lineage was detected in these cells. Our data revealed that the cocktail consisting of DMEM/LG, FBS, RA, FGF, and EGF (DF/R/Fg/E group) induced hUCM cells to express the highest percentage of nestin, ß-tubulin III, neurofilament, and CNPase. The DF/Ds/Fg/E group led to the highest percentage of GFAP expression. While the expression levels of NF, GFAP, and CNPase were the lowest in the DF group. The least percentage of nestin and ß-tubulin III expression was observed in the DF/Ds group. We may conclude that FGF and EGF are important inducers for differentiation of hUCMs into neuron, astrocyte and oligodendrocyte. RA can induce hUCMs to differentiate into neuron and oligodendrocyte while for astrocyte differentiation DMSO had a pivotal role.     

ACPA fine-specificity profiles in early rheumatoid arthritis patients do not correlate with clinical features at baseline or with disease progression

Introduction

Autoantibodies against citrullinated peptides/proteins (ACPA) are found in approximately 75% of the sera of patients with rheumatoid arthritis (RA). The RA-specific ACPA are frequently present prior to disease onset and their presence associates with a more erosive disease course. ACPA can therefore be used to aid the diagnosis and prognosis of RA. Recently, it became clear that ACPA are very heterogeneous, both in an individual patient and among different patients. The aim of this study was to investigate whether clinically meaningful ACPA profiles exist in early RA patients.

Methods

Twenty citrullinated peptides and the corresponding non-citrullinated control peptides were immobilized on microarray sensor chips. Sera from 374 early arthritis patients were analyzed by surface plasmon resonance imaging (iSPR) of biomolecular interactions on the sensor chip.

Results

Cluster analysis of the reactivities with the citrullinated peptides, after subtraction of the reactivities with the corresponding control peptides confirmed the heterogeneity of the ACPA response in RA and revealed 12 distinct ACPA profiles. The association of the 5 most frequent profiles with clinical features at diagnosis and during the disease course was examined, showing no statistically significant associations.

Conclusions

Compared to the detection of ACPA in RA sera by CCP-based assays, ACPA profiling in early arthritis patients did not reveal associations with disease activity and progression scores.     

Preparation of FMD type A87/IRN inactivated vaccine by gamma irradiation and the immune response on guinea pig

FMD is one of the most economically damaging diseases that affect livestock animals. In this study FMD Virus type A87/IRN was multiplied on BHK21 cells. The virus was titrated by TCID50 method, it was 10^7.5/ml. The FMD virus samples were inactivated by gamma ray from 60Co source at −20°C. Safety test was done by IBRS2 monolayer cell culture method, also antigenicity of irradiated and un-irradiated virus samples were studied by Complement Fixation Test. The Dose/Survival curve for irradiated FMD Virus was drawn, the optimum dose range for inactivation of FMDV type A87/IRN and unaltered antigenicity was obtained 40–44 kGy. The inactivated virus samples by irradiation and ethyleneimine (EI) were formulated respectively as vaccine with Al(OH)3 gel and other substances. The vaccines were inoculated to Guinea pigs and the results of Serum Neutralization Test for the normal vaccine and radio-vaccine showed protective titer after 8 months. The potency test of the inactivated vaccines was done, PD50 Value of the vaccines were calculated 7.06 and 5.6 for inactivated vaccine by EI and gamma irradiation respectively.     

Nucleotide sequence of the tcmII-tcmIV region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens and evidence that the tcmN gene encodes a multifunctional cyclase-dehydratase-O-methyl transferase.

Mutations in the tcmII-tcmIV region of the Streptomyces glaucescens chromosome block the C-3 and C-8 O-methylations of the polyketide antibiotic tetracenomycin C (Tcm C). The nucleotide sequence of this region reveals the presence of two genes, tcmN and tcmO, whose deduced protein products display similarity to the hydroxyindole O-methyl transferase of the bovine pineal gland, an enzyme that catalyzes a phenolic O-methylation analogous to those required for the biosynthesis of Tcm C. The deduced product of the tcmN gene also has an N-terminal domain that shows similarity to the putative ActVII and WhiE ORFVI proteins of Streptomyces coelicolor. The tcmN N-terminal domain can be separated from the remainder of the tcmN gene product, and when coupled on a plasmid with the Tcm C polyketide synthase genes (tcmKLM), this domain enables high-level production of an early, partially cyclized intermediate of Tcm C in a Tcm C- null mutant or in a heterologous host (Streptomyces lividans). By analogy to fatty acid biosynthesis, the tcmKLM polyketide synthase gene products are probably sufficient to produce the linear decaketide precursor of Tcm C; thus, the tcmN N-terminal domain is most likely responsible for one or more of the early cyclizations and, perhaps, the attendant dehydrations that lead to the partially cyclized intermediate. The tcmN gene therefore appears to encode a multifunctional cyclase-dehydratase-3-O-methyl transferase. The tcmO gene encodes the 8-O-methyl transferase.     

Oxidative stress induces Z-DNA-binding protein 1–dependent activation of microglia via mtDNA released from retinal pigment epithelial cells

Oxidative stress, inflammation, and aberrant activation of microglia in the retina are commonly observed in ocular pathologies. In glaucoma or age-related macular degeneration, the chronic activation of microglia affects retinal ganglion cells and photoreceptors, respectively, contributing to gradual vision loss. However, the molecular mechanisms that cause activation of microglia in the retina are not fully understood. Here we show that exposure of retinal pigment epithelial (RPE) cells to chronic low-level oxidative stress induces mitochondrial DNA (mtDNA)-specific damage, and the subsequent translocation of damaged mtDNA to the cytoplasm results in the binding and activation of intracellular DNA receptor Z-DNA-binding protein 1 (ZBP1). Activation of the mtDNA/ZBP1 pathway triggers the expression of proinflammatory markers in RPE cells. In addition, we show that the enhanced release of extracellular vesicles (EVs) containing fragments of mtDNA derived from the apical site of RPE cells induces a proinflammatory phenotype of microglia via activation of ZBP1 signaling. Collectively, our report establishes oxidatively damaged mtDNA as an important signaling molecule with ZBP1 as its intracellular receptor in the development of an inflammatory response in the retina. We propose that this novel mtDNA-mediated autocrine and paracrine mechanism for triggering and maintaining inflammation in the retina may play an important role in ocular pathologies. Therefore, the molecular mechanisms identified in this report are potentially suitable therapeutic targets to ameliorate development of ocular pathologies.     

Full Length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons

Background  

Bcl-2 homology domain (BH) 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid), which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 μM).     

Microstructural characterization of the body key scale morphology in six Iranian endemic <Emphasis Type="Italic">Aphanius</Emphasis> species (Cyprinodontidae): Their taxonomic and evolutionary significance

The microstructural characteristics of the body key scales were described for six Iranian endemic Aphanius Nardo, 1827 species. Among them, five species genetically belong to the “Iranian inland and inland-related Aphanius species, IIRAS” group, while A. ginaonis (Holly, 1929) belongs to the “brackish water species” group. General scale shape in the studied Aphanius species was cycloid with the exception of A. ginaonis, which has cycloid scales with few spinous-like structures present in the posterior edge of the scales. Considering phylogenetic relationships of the studied taxa, the most likely explanation for presence of the spinous scales in Aphanius species is the primary existence of these scales in this group. In addition, among the IIRAS group, the most different lepidont was recognized in scales of the A. isfahanensis Hrbek, Keivany and Coad, 2006 (pointed), which is similar to lepidonts in the A. ginaonis. The scales of these species are polygonal with similar typology. The similarity in lepidont morphology between A. ginaonis and A. isfahanensis can explained by (i) the primary existence of the rounded lepidont in this group, and (ii) since A. ginaonis and A. isfahanensis belong to two separate phylogenetic lineages, therefore, similarity in their lepidont morphology could be results of convergent evolution.     

A genetic study on C5-TRAF1 and progression of joint damage in rheumatoid arthritis

IntroductionThe severity of joint damage progression in rheumatoid arthritis (RA) is heritable. Several genetic variants have been identified, but together explain only part of the total genetic effect. Variants in Interleukin-6 (IL-6), Interleukin-10 (IL-10), C5-TRAF1, and Fc-receptor-like-3 (FCRL3) have been described to associate with radiographic progression, but results of different studies were incongruent. We aimed to clarify associations of these variants with radiographic progression by evaluating six independent cohorts.MethodsIn total 5,895 sets of radiographs of 2,493 RA-patients included in six different independent datasets from the Netherlands, Sweden, Spain and North-America were studied in relation to rs1800795 (IL-6), rs1800896 (IL-10), rs2900180 (C5-TRAF1) and rs7528684 (FCRL3). Associations were tested in the total RA-populations and in anti-citrullinated peptide antibodies (ACPA)-positive and ACPA-negative subgroups per cohort, followed by meta-analyses. Furthermore, the associated region C5-TRAF1 was fine-mapped in the ACPA-negative Dutch RA-patients.ResultsNo associations were found for rs1800795 (IL-6), rs1800896 (IL-10) and rs7528684 (FCRL3) in the total RA-population and after stratification for ACPA. Rs2900180 in C5-TRAF1 was associated with radiographic progression in the ACPA-negative population (P-value meta-analysis = 5.85 × 10⁻⁷); the minor allele was associated with more radiographic progression. Fine-mapping revealed a region of 66Kb that was associated; the lowest P-value was for rs7021880 in TRAF1. The P-value for rs7021880 in meta-analysis was 6.35 × 10⁻⁸. Previous studies indicate that the region of rs7021880 was associated with RNA expression of TRAF1 and C5.ConclusionVariants in IL-6, IL-10 and FCRL3 were not associated with radiographic progression. Rs2900180 in C5-TRAF1 and linked variants in a 66Kb region were associated with radiographic progression in ACPA-negative RA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0514-0) contains supplementary material, which is available to authorized users.