An efficient method for isolation of murine bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) have been isolated based on the ability of adherence to plastic surfaces. The potential of these cells to differentiate along multiple lineages is the key to identifying stem cell populations in the absence of molecular markers. Here we describe a homogenous population of MSCs from mouse bone marrow isolated using a relatively straightforward and novel approach. This method is based on the combination of frequent medium change (FMC) and treatment of the primary cultures with trypsin. Cells isolated using this method demonstrated the MSCs characteristics including their ability to differentiate into mesenchymal lineages. MSCs retained the differentiation potentials in expanded cultures up to 10 passages. Isolated MSCs were reactive to the CD44, Sca-1, and CD90 cell surface markers. MSCs were negative for the hematopoietic surface markers such as CD34, CD11b, CD45, CD31, CD106, CD117 and CD135. The data presented in this report indicated that this method can result in efficient isolation of homogenous populations of MSCs from mouse bone marrow.     

Ultraviolet inactivation and photoreactivation of the cholera phage ‘Kappa’

Summary The lysogenic cholera phage, Kappa is some ten to twenty folds more resistant to UV (254 nm) than are most of the T. phages ofE. coli, or the cholera phage PL 163/10, or the hostV. cholerae strain H218 Sm^r, the 37% (D ₃₇) and 10% (D ₁₀) survival doses being 255.8 J/m² and 633.6 J/m² respectively. The UV-irradiated Kappa phages could be photoreactivated in the hostV. cholerae strain H218 Sm^r to a maximum extent of 40%. The removal of the number of lethal hits per phage by the survival-enhancement treatment (photoreactivation) with time followed an exponential relation, the constant probability of removal of lethal hit per unit time being 2.8 × 10^–2 min^–1. The UV-irradiated phages could also be Weigle reactivated in the host strain H218 Sm^r by a small degree, the maximum reactivation factor (ratio of survivals in UV-irradiated and non-irradiated hosts) being 1.50.     

Regeneration of phytoplasma-free <Emphasis Type=Italic>Artemisia roxburghiana</Emphasis> Besser var. purpurascens (Jacq.) Hook. plants using apical meristem culture

We have used apical meristem culture to develop an efficient protocol for reducing phytoplasma infection of Artemisia roxburghiana Besser var. purpurascens (Jacq.) Hook. plants. Shoot tips of different sizes from phytoplasma-infected field-grown plants were treated with 0.1% mercuric chloride (HgCl₂) for 1 min followed by a 30-s exposure to 70% ethanol. The size of the explants significantly influenced the survival frequency and the success of aseptic culture establishment. Sterile explants responded notably to 13.95 μM Kinetin (Kn) and 0.27 μM α-naphthalene acetic acid (NAA), and a maximum of 38 ± 0.87 shoots per explant could be obtained after 6 weeks of incubation. Sub-culturing of the shoot mass after 8 weeks of culture on the previously described medium to 8.88 μM 6-benzyladenine (BA)- and 0.27 μM NAA-containing medium stimulated further multiplication, elongation and growth of each individual regenerant. Efficient rooting was noted after 5 weeks of transfer on half-strength MS medium containing 4.93 μM IBA. Sequential hardening as hydroponic cultures under culture room and glasshouse conditions led to almost 85 and 98% survival of the regenerants upon transfer to pots and field, respectively. Unlike the original A. roxburghiana plants, the plants raised from tissue culture showed a total absence of inherent phytoplasma infection evaluated via inspection of morphological features, PCR and microscopic observations.     

Dopamine receptor genetic polymorphisms and body composition in undernourished pastoralists: An exploration of nutrition indices among nomadic and recently settled Ariaal men of northern Kenya

Background  

Minor alleles of the human dopamine receptor polymorphisms, DRD2/TaqI A and DRD4/48 bp, are related to decreased functioning and/or numbers of their respective receptors and have been shown to be correlated with body mass, height and food craving. In addition, the 7R minor allele of the DRD4 gene is at a higher frequency in nomadic compared to sedentary populations. Here we examine polymorphisms in the DRD2 and DRD4 genes with respect to body mass index (BMI) and height among men in two populations of Ariaal pastoralists, one recently settled (n = 87) and the other still nomadic (n = 65). The Ariaal live in northern Kenya, are chronically undernourished and are divided socially among age-sets.     

Supramolecular structures of peptide assemblies in membranes by neutron off-plane scattering: method of analysis

In a previous paper (Yang et al., Biophys. J. 75:641-645, 1998), we showed a simple, efficient method of recording the diffraction patterns of supramolecular peptide assemblies in membranes where the samples were prepared in the form of oriented multilayers. Here we develop a method of analysis based on the diffraction theory of two-dimensional liquids. Gramicidin was used as a prototype model because its pore structure in membrane in known. At full hydration, the diffraction patterns of alamethicin and magainin are similar to gramicidin except in the scale of q (the momentum transfer of scattering), clearly indicating that both alamethicin and magainin form pores in membranes but of different sizes. When the hydration of the multilayer samples was decreased while the bilayers were still fluid, the in-plane positions of the membrane pores became correlated from one bilayer to the next. We believe that this is a new manifestation of the hydration force. The effect is most prominent in magainin patterns, which are used to demonstrate the method of analysis. When magainin samples were further dehydrated or cooled, the liquid-like diffraction turned into crystal-like patterns. This discovery points to the possibility of investigating the supramolecular structures with high-order diffraction.     

DRAGON, a bone morphogenetic protein co-receptor

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)beta superfamily of ligands that regulate many crucial aspects of embryonic development and organogenesis. Unlike other TGFbeta ligands, co-receptors for BMP ligands have not been described. Here we show that DRAGON, a glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, which is expressed early in the developing nervous system, enhances BMP but not TGFbeta signaling. DRAGON binds directly to BMP2 and BMP4 but not to BMP7 or other TGFbeta ligands. The enhancing action of DRAGON on BMP signaling is also reduced by administration of Noggin, a soluble BMP antagonist, indicating that the action of DRAGON is ligand-dependent. DRAGON associates directly with BMP type I (ALK2, ALK3, and ALK6) and type II (ActRII and ActRIIB) receptors, and its signaling is reduced by dominant negative Smad1 and ALK3 or -6 receptors. In the Xenopus embryo, DRAGON both reduces the threshold of the ability of Smad1 to induce mesodermal and endodermal markers and alters neuronal and neural crest patterning. The direct interaction of DRAGON with BMP ligands and receptors indicates that it is a BMP co-receptor that potentiates BMP signaling.     

Hydrogeochemistry of seasonal variation of Urmia Salt Lake, Iran

Urmia Lake has been designated as an international park by the United Nations. The lake occupies a 5700 km2 depression in northwestern Iran. Thirteen permanent rivers flow into the lake. Water level in the lake has been decreased 3.5 m in the last decade due to a shortage of precipitation and progressively dry climate. Geologically the lake basin is considered to be a graben of tectonic origin. Na, K, Ca, Li and Mg are the main cations with Cl, SO4, and HCO3 as the main anions. F & Br are the other main elements in the lake. A causeway crossing the lake is under construction, which may affect the lake's annual geochemistry. The main object of this project is mainly to consider the potential of K-mineral production along with ongoing salt production. Seven hundred and four samples were taken and partially analyzed for the main cations and anions. Surface water (0.5 m. depth) was analyzed for Na, K, Mg, Ca, Br and Li, and averaged 87.118 g/lit, 1.48 g/lit, 4.82 g/lit, 4.54 g/lit, 1.19 ppm and 12.7 ppm respectively for the western half of the lake. Sodium ranged between 84 to 91.2 g/lit, and showed higher concentrations in the south than in the north. This unexpected result may be caused by shallower depth in the south and a higher net evaporation effect. Calcium ranged between 4.2 to 5 g/lit, apparently slightly higher in the north. K is higher in the south, possibly due to rivers entering from south that may carry slightly higher K in solution. In the middle-range samples (0.5–5 m.), K averaged 1.43 g/lit and ranged from 1.40 to 1.46 g/lit. At this intermediate depth the distribution of K is clearly higher to the south of the causeway that is currently under construction. It is not clear whether this increase is the effect of the causeway or the effect of the salty Aji-Chay River to the east, and the Khoy salt domes to the north of the lake. At depth (5 m–10 m), K averaged 1.48 g/lit and ranged from 1.4 to 1.49 g/lit, differing only in the second decimal from the average of the middle and surface samples. Ignoring the small difference between the averages of the three sample depths, the distribution of K is highly homogeneous in the lake water due to the mixing process. Therefore causeway construction has not yet strongly affected K distribution, or it may be at the starting point. Magnesium concentration ranged from 4.6 to 5-g/lit, and was elevated in the south. This differs somewhat compared to calcium. Lithium, with an average of 12–13 ppm, is slightly higher in the south, and has not shown any significant variation in all three seasons. Iodine was below the detection limit in the lake. Urmia Lake, geochemically, is highly uniform both to the south and north of the causeway, in both the surface and deep brines. K and Mg, which average 1.48 and 6.6 g/lit in order, could be elements worth production in addition to the NaCl currently being produced from the lake. Br, F, Li and B in the limit of <50 ppm don't look to be in the economical range.     

Mesenchymal stem cells promote alveolar epithelial cell wound repair in vitro through distinct migratory and paracrine mechanisms

Background

Mesenchymal stem cells (MSC) are in clinical trials for widespread indications including musculoskeletal, neurological, cardiac and haematological disorders. Furthermore, MSC can ameliorate pulmonary fibrosis in animal models although mechanisms of action remain unclear. One emerging concept is that MSCs may have paracrine, rather than a functional, roles in lung injury repair and regeneration.

Methods

To investigate the paracrine role of human MSC (hMSC) on pulmonary epithelial repair, hMSC-conditioned media (CM) and a selected cohort of hMSC-secretory proteins (identified by LC-MS/MS mass spectrometry) were tested on human type II alveolar epithelial cell line A549 cells (AEC) and primary human small airway epithelial cells (SAEC) using an in vitro scratch wound repair model. A 3D direct-contact wound repair model was further developed to assess the migratory properties of hMSC.

Results

We demonstrate that MSC-CM facilitates AEC and SAEC wound repair in serum-dependent and –independent manners respectively via stimulation of cell migration. We also show that the hMSC secretome contains an array of proteins including Fibronectin, Lumican, Periostin, and IGFBP-7; each capable of influencing AEC and SAEC migration and wound repair stimulation. In addition, hMSC also show a strong migratory response to AEC injury as, supported by the observation of rapid and effective AEC wound gap closure by hMSC in the 3D model.

Conclusion

These findings support the notion for clinical application of hMSCs and/or their secretory factors as a pharmacoregenerative modality for the treatment of idiopathic pulmonary fibrosis (IPF) and other fibrotic lung disorders.     

Timing of enteral feeding in cerebral malaria in resource-poor settings: a randomized trial

Background

Early start of enteral feeding is an established treatment strategy in intubated patients in intensive care since it reduces invasive bacterial infections and length of hospital stay. There is equipoise whether early enteral feeding is also beneficial in non-intubated patients with cerebral malaria in resource poor settings. We hypothesized that the risk of aspiration pneumonia might outweigh the potential benefits of earlier recovery and prevention of hypoglycaemia.

Method and Findings

A randomized trial of early (day of admission) versus late (after 60 hours in adults or 36 hours in children) start of enteral feeding was undertaken in patients with cerebral malaria in Chittagong, Bangladesh from May 2008 to August 2009. The primary outcome measures were incidence of aspiration pneumonia, hypoglycaemia and coma recovery time. The trial was terminated after inclusion of 56 patients because of a high incidence of aspiration pneumonia in the early feeding group (9/27 (33%)), compared to the late feeding group (0/29 (0%)), p = 0.001). One patient in the late feeding group, and none in the early group, had hypoglycaemia during admission. There was no significant difference in overall mortality (9/27 (33%) vs 6/29 (21%), p = 0.370), but mortality was 5/9 (56%) in patients with aspiration pneumonia.

Conclusions

In conclusion, early start of enteral feeding is detrimental in non-intubated patients with cerebral malaria in many resource-poor settings. Evidence gathered in resource rich settings is not necessarily transferable to resource-poor settings.

Trial Registration

Controlled-Trials.com ISRCTN57488577     

Effects of 16 genetic variants on fasting glucose and type 2 diabetes in South Asians: ADCY5 and GLIS3 variants may predispose to type 2 diabetes

BackgroundThe Meta-Analysis of Glucose and Insulin related traits Consortium (MAGIC) recently identified 16 loci robustly associated with fasting glucose, some of which were also associated with type 2 diabetes. The purpose of our study was to explore the role of these variants in South Asian populations of Punjabi ancestry, originating predominantly from the District of Mirpur, Pakistan.Conclusions/SignificanceAlthough only the SLC30A8 rs11558471 SNP was nominally associated with fasting glucose in our study, the finding that 12 out of 16 SNPs displayed a direction of effect consistent with European studies suggests that a number of these variants may contribute to fasting glucose variation in individuals of South Asian ancestry. We also provide evidence for the first time in South Asians that alleles of SNPs in GLIS3 and ADCY5 may confer risk of type 2 diabetes.