Rapid Detection of Senescent Mesenchymal Stromal Cells by a Fluorescent Probe

Despite intense interest in human mesenchymal stromal cells (MSCs), monitoring of the progressive occurrence of senescence has been hindered by the lack of efficient detection tools. Here, the discovery of a novel MSC senescence‐specific fluorescent probe (CyBC9) identified by a high‐throughput screen is reported. Compared with the prototypical senescence‐associated β‐galactosidase (SA‐β‐gal) staining, the CyBC9 assay is rapid (2 h) and nontoxic and can thus be applied to live cells. It is shown that CyBC9 is able to stain early and late senescent populations both in monolayer‐ and in microcarrier‐based cultures. Finally, to investigate the mechanism of CyBC9, colocalization assays are performed and it is found that CyBC9 is accumulated in the mitochondria of senescent MSCs presumably due to the loss of membrane potential. Taken together, it is expected that CyBC9 will be a useful tool to ameliorate cell therapy through rapid and early screening of senescent phenotypes in clinically relevant MSCs.     

Wheat tandem kinases provide insights on disease‐resistance gene flow and host–parasite co‐evolution

Stripe (yellow) rust, caused by the fungus Puccinia striiformis f. sp. tritici (Pst), is a destructive disease of wheat spread globally. Wild emmer wheat (Triticum turgidum ssp. dicoccoides; WEW) is known as a source for novel Pst resistance genes (R‐gene), but our knowledge on wheat‐Pst co‐evolution in natural populations is limited. Yr15 is a WEW (accession G25) gene, which confers a broad‐spectrum resistance to Pst, and encodes a tandem kinase‐pseudokinase protein designated as WTK1. Exon–intron comparisons of multiple WTK1 homoeologous and paralogous copies scattered in allopolyploid wheat genomes enabled us to develop functional molecular markers (FMMs), which were used for population genetic study. The functional allele (Wtk1) was absent in a worldwide collection of 513 wheat cultivars, except for 32 introgression lines with Yr15 from G25, as well as in 84% of the 382 tested WEW accessions collected across the Fertile Crescent. Yr15 was found to be distributed along a narrow axis from Mt Carmel to the Anti‐Lebanon Mountains ridge, mostly at elevations above c. 500 m, where the climatic conditions are favorable for disease development, therefore providing insights on gene flow and host–parasite co‐evolution in WEW natural habitats. Moreover, the worldwide absence of Wtk1 in cultivated wheat and in WEW natural populations from southeast Turkey, where wheat is believed to have been domesticated, proposes that Yr15 was rather left behind, than lost during domestication. Our results highlight the importance of conservation of WEW populations in their natural habitats for discovery of novel R‐genes and studies of host–parasite co‐evolution.     

Diarrheal Illness and Healthcare Seeking Behavior among a Population at High Risk for Diarrhea in Dhaka,Bangladesh

Diarrhea remains one of the major causes of death in Bangladesh. We studied diarrheal disease risk and healthcare seeking behavior among populations at high risk for diarrhea in Dhaka, Bangladesh. Data were obtained from a cross-sectional survey conducted during April and September 2010. The prevalence of diarrhea was calculated by age-group and sex. A generalized estimating equation with logit link function was used to predict diarrheal disease risk and seeking care from a professional healthcare provider. Of 316,766 individuals, 10% were young children (<5 years). The prevalence of diarrhea was 16 per 1000 persons among all ages; young children accounted for 44 per 1000 persons. Prevalence of diarrhea was significantly higher (p=.003) among younger males (<15 years) compared to that among younger females. In contrast, prevalence of diarrhea was significantly higher (p<.0001) among older females (≥15 years) compared to that among older males. An increased risk for diarrhea was observed in young children, males, and those staying in rented houses, lower family members in the house, using non-sanitary toilets, living in the area for short times, living in a community with less educated persons, living in a community with less use of safe water source for drinking, or living close to the hospital. About 80% of those with diarrhea sought care initially from a non-professional healthcare provider. Choice of the professional healthcare provider was driven by age of the patient, educational status of the household head, and hygienic practices by the household. The study reaffirms that young children are at greater risk for diarrhea. Like other developing countries most people in this impoverished setting of Dhaka are less likely to seek care from a professional healthcare provider than from a non-professional healthcare provider, which could be attributed to a higher number of diarrheal deaths among young children in Bangladesh. Dissemination of information on health education, increasing the supply of skilled healthcare providers, and low-cost and quality healthcare services may encourage more people to seek care from professional healthcare providers, thus may help reduce child mortality in the country. Further studies are warranted to validate the results.     

Parallel microgeographic patterns of genetic diversity and divergence revealed by allozyme, RAPD, and microsatellites in Triticum dicoccoides at Ammiad, Israel

The levels of genetic diversity were compared by means of 35 allozyme, 60 RAPD, and 25 microsatellite (SSR) markers for 75–175 individuals of tetraploid wild emmer wheat (Triticum dicoccoides) collected in 1993 from a microgeographic microsite, Ammiad, north of the Sea of Galilee, Israel. This microsite included four major habitats, which showed highly significant differentiation in ecological factors, in particular with respect to rock cover, proximity and height, and surface soil moisture in the early growing season of T. dicoccoides. Higher within-subpopulation genetic diversity was found in the primarily non-coding DNA regions (RAPD and SSR) rather than in the protein-coding (allozymes) regions. However, much larger gene differentiation (G _ST) among the subpopulations was observed in the protein-coding allozymes than in the RAPDs and SSRs. Larger genetic distance was found at SSR loci, followed by allozyme and RAPD loci. The subpopulations in drier habitats tend to have higher allozyme, RAPD and SSR diversities (He), the relatively wet Karst subpopulation showed only about half He of the other relatively drier habitats. The subpopulations with larger difference of soil moisture between habitats tend to show larger genetic distances at allozyme, RAPD and SSR loci. These results suggest that climatic selection through aridity stress may be an important factor acting on both structural protein-coding and presumably partly regulatory non-coding DNA regions, resulting in microscale adaptive patterns, although hitchhiking and random drift may also intervene. These results have profound implications for genetic conservation both in situ and ex situ.     

Construction and characterization of a half million clone BAC library of durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> ssp. <Emphasis Type="Italic">durum</Emphasis>)

Durum wheat (Triticum turgidum ssp. durum, 2n = 4x = 28, genomes AB) is an economically important cereal used as the raw material to make pasta and semolina. In this paper we present the construction and characterization of a bacterial artificial chromosome (BAC) library of tetraploid durum wheat cv. Langdon. This variety was selected because of the availability of substitution lines that facilitate the assignment of BACs to the A and B genome. The selected Langdon line has a 30-cM segment of chromosome 6BS from T. turgidum ssp. dicoccoides carrying a gene for high grain protein content, the target of a positional cloning effort in our laboratory. A total of 516,096 clones were organized in 1,344 384-well plates and blotted on 28 high-density filters. Ninety-eight percent of these clones had wheat DNA inserts (0.3% chloroplast DNA, 1.4% empty clones and 0.3% empty wells). The average insert size of 500 randomly selected BAC clones was 131 kb, resulting in a coverage of 5.1-fold genome equivalents for each of the two genomes, and a 99.4% probability of recovering any gene from each of the two genomes of durum wheat. Six known copy-number probes were used to validate this theoretical coverage and gave an estimated coverage of 5.8-fold genome equivalents. Screening of the library with 11 probes related to grain storage proteins and starch biosynthesis showed that the library contains several clones for each of these genes, confirming the value of the library in characterizing the organization of these important gene families. In addition, characterization of fingerprints from colinear BACs from the A and B genomes showed a large differentiation between the A and B genomes. This library will be a useful tool for evolutionary studies in one of the best characterized polyploid systems and a source of valuable genes for wheat. Clones and high-density filters can be requested at Communicated by P. LangridgeThe first two authors contributed equally to the investigation     

New aspects of the regulation of glycosphingolipid receptor function

We propose that the fatty acid heterogeneity of glycosphingolipids may compensate for the relative few and simple glycosphingolipid structures found in mammalian cells. Variation in GSL fatty acid composition may mediate aglycone regulation of GSL membrane receptor function by a differential interaction with cholesterol and other membrane components which may be differentially organized within plasma membrane lipid domains. These concepts are specifically illustrated in model membrane studies and in relation to the role of the glycolipid, globotriaosyl ceramide (Gb₃) in verotoxin-induced renal pathology and gp120 binding in HIV infection.     

Formulation,Characterization, and Clinical Evaluation of Microemulsion Containing Clotrimazole for Topical Delivery

The objective of the present study was to formulate and evaluate microemulsion systems for topical delivery of clotrimazole (CTM). The solubility of CTM in various oils was determined to select the oil phase of the microemulsion systems. Pseudoternary phase diagrams were constructed to identify the area of microemulsion existence. Five CTM microemulsion formulations (M1–M5) were prepared and evaluated for their thermodynamic stability, pH, refractive index, droplet size, viscosity, and in vitro release across cellulose membrane. Among the prepared microemulsion formulations, M3 (lemon oil/Tween 80/n-butanol/water) and M4 (isopropyl myristate/Tween 80/n-butanol/water) microemulsion systems were found to be promising according to their physical properties and CTM cumulative percentage release. Gel form of M3 and M4 were prepared using 1% Carbopol 940 as the hydrogel matrix. Both formulations were evaluated in the liquid and gel forms for drug retention in the skin in comparison to the marketed CTM topical cream and their stability examined after storage at 40°C for 6 months. Microemulsion formulations achieved significantly higher skin retention for CTM over the CTM cream. Stability studies showed that M4 preparations were more stable than M3. The in vitro anti-fungal activity of M4 against Candida albicans was higher than that of the conventional cream. Moreover, clinical evaluation proved the efficacy and tolerability of this preparation in the treatment of various topical fungal infections.     

A Physical Map of the Short Arm of Wheat Chromosome 1A

Bread wheat (Triticum aestivum) has a large and highly repetitive genome which poses major technical challenges for its study. To aid map-based cloning and future genome sequencing projects, we constructed a BAC-based physical map of the short arm of wheat chromosome 1A (1AS). From the assembly of 25,918 high information content (HICF) fingerprints from a 1AS-specific BAC library, 715 physical contigs were produced that cover almost 99% of the estimated size of the chromosome arm. The 3,414 BAC clones constituting the minimum tiling path were end-sequenced. Using a gene microarray containing ∼40 K NCBI UniGene EST clusters, PCR marker screening and BAC end sequences, we arranged 160 physical contigs (97 Mb or 35.3% of the chromosome arm) in a virtual order based on synteny with Brachypodium, rice and sorghum. BAC end sequences and information from microarray hybridisation was used to anchor 3.8 Mbp of Illumina sequences from flow-sorted chromosome 1AS to BAC contigs. Comparison of genetic and synteny-based physical maps indicated that ∼50% of all genetic recombination is confined to 14% of the physical length of the chromosome arm in the distal region. The 1AS physical map provides a framework for future genetic mapping projects as well as the basis for complete sequencing of chromosome arm 1AS.     

A consensus framework map of durum wheat (Triticum durum Desf.) suitable for linkage disequilibrium analysis and genome-wide association mapping

Background

Durum wheat (Triticum durum Desf.) is a tetraploid cereal grown in the medium to low-precipitation areas of the Mediterranean Basin, North America and South-West Asia. Genomics applications in durum wheat have the potential to boost exploitation of genetic resources and to advance understanding of the genetics of important complex traits (e.g. resilience to environmental and biotic stresses). A dense and accurate consensus map specific for T. durum will greatly facilitate genetic mapping, functional genomics and marker-assisted improvement.

Results

High quality genotypic data from six core recombinant inbred line populations were used to obtain a consensus framework map of 598 simple sequence repeats (SSR) and Diversity Array Technology^® (DArT) anchor markers (common across populations). Interpolation of unique markers from 14 maps allowed us to position a total of 2,575 markers in a consensus map of 2,463 cM. The T. durum A and B genomes were covered in their near totality based on the reference SSR hexaploid wheat map. The consensus locus order compared to those of the single component maps showed good correspondence, (average Spearman’s rank correlation rho ρ value of 0.96). Differences in marker order and local recombination rate were observed between the durum and hexaploid wheat consensus maps. The consensus map was used to carry out a whole-genome search for genetic differentiation signatures and association to heading date in a panel of 183 accessions adapted to the Mediterranean areas. Linkage disequilibrium was found to decay below the r² threshold = 0.3 within 2.20 cM, on average. Strong molecular differentiations among sub-populations were mapped to 87 chromosome regions. A genome-wide association scan for heading date from 27 field trials in the Mediterranean Basin and in Mexico yielded 50 chromosome regions with evidences of association in multiple environments.

Conclusions

The consensus map presented here was used as a reference for genetic diversity and mapping analyses in T. durum, providing nearly complete genome coverage and even marker density. Markers previously mapped in hexaploid wheat constitute a strong link between the two species. The consensus map provides the basis for high-density single nucleotide polymorphic (SNP) marker implementation in durum wheat.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-873) contains supplementary material, which is available to authorized users.