Culture and neural differentiation of rat bone marrow mesenchymal stem cells in vitro

Adult bone marrow mesenchymal stem cells (MSCs) can differentiate into several types of mesenchymal cells, including osteocytes, chondrocytes, and adipocytes, but can also differentiate into non-mesenchymal cells, such as neural cells, under appropriate experimental conditions. Until now, many protocols for inducing neuro-differentiation in MSCs in vitro have been reported. But due to the differences in MSCs' isolation and culture conditions, the results of previous studies lacked consistency and comparability. In this study, we induced differentiation into neural phenotype in the same MSCs population by three different treatments: beta-mercaptoethanol, serum-free medium and co-cultivation with fetal mouse brain astrocytes. In all of the three treatments, MSCs could express neural markers such as NeuN or GFAP, associating with remarkable morphological modifications. But these treatments led to neural phenotype in a non-identical manner. In serum-free medium, MSCs mainly differentiated into neuron-like cells, expressing neuronal marker NeuN, and BME can promote this process. Differently, after co-culturing with astrocytes, MSCs leaned to differentiate into GFAP(+) cells. These data confirmed that MSCs can exhibit plastic neuro-differentiational potential in vitro, depending on the protocols of inducement.     

Effects of substratum on the diversity and stability of ammonia-oxidizing communities in a constructed wetland used for wastewater treatment

AIM: To study the relationship between the nature of the substratum and the diversity and stability of the ammonia-oxidizing microbial community in a constructed wetland for the treatment of wastewaters. METHODS AND RESULTS: Samples have been taken the year around from sections of the wetland filled with different substrata. When present, the root zones of the helophyte Phragmites australis were also sampled. The diversity of the ammonia-oxidizing community was established by a coupled PCR-DGGE method based on the 16s rRNA gene. Averaged over the seasons, no large differences in community composition were observed between the different substrata, although the section with zeolite always showed the highest frequencies of bands belonging to ammonia-oxidizing bacteria of the beta-subclass of the Proteobacteria. Only sequences related to the Nitrosospira lineage were detected. Averaged again over the seasons, the section with zeolite was also most constant with respect to the potential ammonia-oxidizing activity. CONCLUSIONS: Although the ammonia-oxidizing communities did not differ significantly between the different sections of the constructed wetland, the characteristics of zeolite were most appropriate to accommodate a stable and active community of ammonia-oxidizing bacteria. The presence of the helophyte had no effect on the diversity and stability of the ammonia-oxidizing community. SIGNIFICANCE AND IMPACT OF THE STUDY: It has been shown that substrata used in constructed wetlands made no distinction between ammonia-oxidizing strains in relation to attachment. However, zeolite had the best performance with respect to activity over the seasons.     

Enhancement of Sodium Current in NG108-15 Cells during Neural Differentiation is Mainly due to an Increase in NaV1.7 Expression

It is well known that morphological and functional changes during neural differentiation sometimes accompany the expression of various voltage-gated ion channels. In this work, we investigated whether the enhancement of sodium current in differentiated neuroblastoma × glioma NG108-15 cells treated with dibutyryl cAMP is related to the expression of voltage-gated sodium channels. The results were as follows. (1) Sodium current density on peak voltage in differentiated cells was significantly enhanced compared with that in undifferentiated cells, as detected by the whole-cell patch clamp method. The steady-state inactivation curve in differentiated cells was similar to that for undifferentiated cells, but a hyperpolarized shift in the activation curve for differentiated cells was observed. The sodium currents of differentiated and undifferentiated cells were completely inhibited by 10⁻⁷ M tetrodotoxin (TTX). (2) The only Na_V mRNA with an increased expression level during neuronal differentiation was that for Na_V1.7, as observed by real-time PCR analysis. (3) The increase in the level of Na_V1.7 α subunit expression during neuronal differentiation was also observed by immunocytochemistry; in particular, the localization of Na_V1.7 α subunits on the soma, varicosities and growth cone was significant. These results suggest that the enhancement of TTX-sensitive sodium current density in differentiated NG108-15 cells is mainly due to the increase in the expression of the TTX-sensitive voltage-gated Na⁺ channel, Na_V1.7.     

Prospectors' colony attendance is sex-specific and increases future recruitment chances in a seabird

In most long-lived vertebrates, including seabirds, young non-breeders often attend potential breeding sites. In seabird colonies, this prospecting behaviour has nearly never been studied with respect to potential sex-specific benefits, and its fitness consequences are largely unknown. We compared attendance patterns of sexed common tern prospectors at six subcolonies with future breeding status and nesting site choice. We also tested for potential effects of population density. Birds that arrived earlier at the colony were recorded more often along the season. This arrival effect was stronger in males, which generally spent more time at the colony. Birds prospecting for two consecutive years attended the colony more intensively in the second year. A high colony attendance enhanced recruitment probability in both sexes, but only in females, it was linked with a higher probability to return. Attendance at a preferred subcolony increased during the season. For first breeding, individuals favoured the subcolony where they had prospected most intensively in the previous season. In males, this subcolony fidelity was stronger and increased simultaneously to breeding pair density. We conclude that prospecting is a process of integration into the community of breeders, and that benefits are higher for males, the more territorial sex in this species.     

溶血磷脂酸对离体培养的大鼠胚胎神经干细胞向神经胶质细胞分化的影响

本研究用免疫细胞化学荧光双标技术观察了溶血磷脂酸（lysophosphatidic acid,LPA）对大鼠胚胎神经干细胞（neural stem cells,NSCs）分化为少突胶质细胞（galactocerebroside—positive,Gal-C阳性）和星形胶质细胞（grim fibrillary acidic protein-positive,GFAP阳性）的影响,并且用RT-PCR技术对NSCs可能表达的LPA受体进行分析。结果显示：（1）加入不同浓度（0．010．0μmol／L）LPA,第7天进行检测时,少突胶质细胞数量呈明显的剂量依赖性增加,峰值出现在1．0μmol／LLPA组,少突胶质细胞所占百分比从对照组的8．5％增加到32．6％;（2）星形胶质细胞的分化几乎不受LPA的影响,第7天时各LPA处理组星形胶质细胞百分比与对照组相比均无显著性差异;（3）RT-PCR结果显示,大鼠胚胎NSCs的LPA1和LPA3受体表达明显,而LPA3受体表达很弱。以上结果表明,较低浓度的LPA可能作为细胞外信号,通过LPA1和LPA3受体促进大鼠胚胎NSCs向少突胶质细胞分化和生成,但对星形胶质细胞的分化过程无明显影响。     

MicroRNA-223 is a key factor in osteoclast differentiation

MicroRNAs (miRNAs) are a class of noncording RNAs that control gene expression by translational inhibition and messenger RNAs (mRNAs) degradation in plants and animals. Although miRNAs have been implicated in developmental and homeostatic events of vertebrates and invertebrates, the role of miRNAs in bone metabolism has not been explored. Here, we show that microRNA-223 (miR-223) is expressed in RAW264.7 cells, mouse osteoclast precursor cell lines, and plays a critical role in osteoclast differentiation. We constructed miR-223 short interfering RNA (siRNA) or precursor miR-223 (pre-miR-223) overexpression retroviral vectors, and established miR-223 knockdown by siRNA or pre-miR-223 overexpression in stably infected RAW264.7 cells. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells were observed in miR-223 knockdown cells as well as control cells. In contrast, pre-miR-223 overexpression completely blocked TRAP-positive multinucleated cell formation compared with control cells. Apoptotic cells were not observed in this study. Our results indicate that miR-223 plays an essential role during osteoclast differentiation, and miR-223 might be a viable therapeutic target for a range of bone metabolic disorders with excess osteoclast activity.     

VDR-mediated inhibition of DKK1 and SFRP2 suppresses adipogenic differentiation of murine bone marrow stromal cells

Osteoblasts and adipocytes are thought to derive from a common bone marrow stromal cell (BMSC) precursor. Activation of the canonical Wnt signaling pathway plays a pivotal role in the differentiation of BMSCs along either of these two lineages, promoting osteogenesis and inhibiting adipogenesis. Liganded nuclear receptors, including the vitamin D receptor (VDR) and peroxisomal proliferator-activated receptor gamma (PPARgamma), can also affect BMSCs differentiation. To address whether VDR ablation modulates the differentiation of BMSCs into the osteoblast or adipogenic lineages, BMSCs were isolated from VDR null mice and from their wild-type littermates. VDR ablation did not alter osteoblastic differentiation. However, when cultured under adipogenic conditions, BMSCs from the VDR null mice expressed higher mRNA levels of PPARgamma and of markers of adipogenic differentiation. An increase in the size and number of mature adipocyte foci was also observed in cultures isolated from VDR null mice relative to those isolated from wild-type mice. To address whether the increased adipogenesis observed in the VDR null cultures was associated with inhibition of the canonical Wnt signaling pathway, mRNA levels for DKK1 and SFRP2 were examined. Cultures from the VDR null mice expressed higher levels of mRNA encoding DKK1 and SFRP2 than did the wild-type cultures. This difference is, at least in part, due to ligand-dependent actions of the VDR, since 1,25-dihydroxyvitamin D3 suppressed DKK1 and SFRP2 expression in wild-type cultures. Thus, the VDR inhibits adipogenesis of BMSCs at least in part by suppressing the expression of inhibitors of the canonical Wnt signaling pathway.     

Microsatellite null alleles and estimation of population differentiation

Microsatellite null alleles are commonly encountered in population genetics studies, yet little is known about their impact on the estimation of population differentiation. Computer simulations based on the coalescent were used to investigate the evolutionary dynamics of null alleles, their impact on F(ST) and genetic distances, and the efficiency of estimators of null allele frequency. Further, we explored how the existing method for correcting genotype data for null alleles performed in estimating F(ST) and genetic distances, and we compared this method with a new method proposed here (for F(ST) only). Null alleles were likely to be encountered in populations with a large effective size, with an unusually high mutation rate in the flanking regions, and that have diverged from the population from which the cloned allele state was drawn and the primers designed. When populations were significantly differentiated, F(ST) and genetic distances were overestimated in the presence of null alleles. Frequency of null alleles was estimated precisely with the algorithm presented in Dempster et al. (1977). The conventional method for correcting genotype data for null alleles did not provide an accurate estimate of F(ST) and genetic distances. However, the use of the genetic distance of Cavalli-Sforza and Edwards (1967) corrected by the conventional method gave better estimates than those obtained without correction. F(ST) estimation from corrected genotype frequencies performed well when restricted to visible allele sizes. Both the proposed method and the traditional correction method have been implemented in a program that is available free of charge at http://www.montpellier.inra.fr/URLB/. We used 2 published microsatellite data sets based on original and redesigned pairs of primers to empirically confirm our simulation results.     

Androgens drive divergent responses to salt stress in male versus female rat kidneys

Dahl-Iwai (DI) salt-sensitive rats were studied using microarrays to identify sex-specific differences in the kidney, both basal differences and differences in responses to a high-salt diet. In DI rat kidneys, gene expression profiles demonstrated inflammatory and fibrotic responses selectively in females. Gonadectomy of DI rats abrogated sex differences in gene expression. Gonadectomized female and gonadectomized male DI rats both responded to high salt with the same spectrum of gene expression changes as intact female DI rats. Androgens dominated the sex-selective responses to salt. Several androgen-responsive genes with roles potentiating the differential responses to salt were identified, including increased male expression of angiotensin-vasopressin receptor and prolactin receptor, decreased 5 alpha-reductase, and mixed increases and decreases in expression of Cyp4a genes that can produce eicosanoid hormones. These sex differences potentiate sodium retention by males and increase kidney function during gestation in females.